Administration and dosage of diaminophenothiazines

ABSTRACT

The invention provides novel regimens for treatment of neurodegenerative disorders utilising methylthioninium (MT)-containing compounds. The regimens are based on novel findings in relation to the dosage of MT compounds, and their interaction with symptomatic treatments based on modulation of acetylcholinesterase levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation application of U.S. application Ser.No. 16/320,148, filed Jan. 24, 2019, which is a National Stage filingunder 35 U.S.C. 371 of International Patent Application Serial No.PCT/EP2017/068749, filed Jul. 25, 2017, which claims priority to GreatBritain Application No. 1612863.9, filed Jul. 25, 2016, and GreatBritain Application No. 1710382.1, filed Jun. 29, 2017. The contents ofthese applications are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates generally to methods and materials for usein the treatment or prophylaxis of diseases of protein aggregation, forexample cognitive disorders, using diaminophenothiazines.

BACKGROUND ART

Aberrant protein aggregation is believed to be a proximal cause ofnumerous disease states, which may be manifested as neurodegeneration,clinical dementia, and other pathological symptoms.

In general, the aberrant protein aggregation is that which arises froman induced conformational polymerisation interaction, i.e., one in whicha conformational change of the protein, or in a fragment thereof, givesrise to templated binding and aggregation of further (precursor) proteinmolecules in a self-propagating manner.

Once nucleation is initiated, an aggregation cascade may ensue whichinvolves the induced conformational polymerisation of further proteinmolecules, leading to the formation of toxic product fragments inaggregates which are substantially resistant to further proteolysis.

For example certain conditions of dementia may be characterised by aprogressive accumulation of intracellular and/or extracellular depositsof proteinaceous structures such as β-amyloid plaques andneurofibrillary tangles (NFTs) in the brains of affected patients. Theappearance of these lesions largely correlates with pathologicalneurofibrillary degeneration and brain atrophy, as well as withcognitive impairment (see, e.g., Mukaetova-Ladinska, E. B. et al., 2000,Am. J. Pathol., Vol. 157, No. 2, pp. 623-636).

Current approved treatments for Alzheimer's disease includeacetylcholinesterase inhibitors (AChEIs) and the N-methyl-D-aspartatereceptor antagonist memantine. These are symptomatic and do not addressthe underlying disease pathology. Therapies targeting the amyloidpathology have so far proved unsuccessful in late stage clinical trials(Geerts et al., 2013; Mullane and Williams, 2013). According to a recentLancet Neurology Commission, “an effective treatment for AD is perhapsthe greatest unmet medical need facing modern medicine”, (Winblad etal., 2016) not least because the global economic cost of dementia isestimated to be $818 billion, or 0.65% of global gross domestic product(Alzheimer's Disease International, 2015).

NFTs (the pathology discovered by Alois Alzheimer, (Alzheimer, 1907))are made up of paired helical filaments (PHFs), composed predominantlyof a 12-kDa repeat-domain fragment of the microtubule-associated proteintau (Wischik et al., 1985; Wischik et al., 1988a,b). Numerous studieshave confirmed a quantitative link for the spread of neurofibrillarytangle pathology and the quantity of aggregated tau with both the extentof clinical dementia and functional molecular imaging deficits inAlzheimer's disease (Arriagada et al., 1992; Brier et al., 2016;Giannakopoulos et al., 2003; Josephs et al., 2003; Maruyama et al.,2013). Since pathological aggregation of tau protein begins at least 20years prior to any of the clinical manifestations, (Braak and delTredici, 2013) targeting this pathology offers a rational approach toboth treatment and prevention of AD and related tau aggregationdisorders (Huang and Mucke, 2012; Wischik et al., 2014; Wischik et al.,2010).

The tau fragment originally identified as an intrinsic structuralconstituent of the PHF core has prion-like properties in vitro in thatit captures normal tau protein with very high affinity (Lai et al.,2016) and converts it to a proteolytically stable replicate of itself(Wischik et al., 1996; Harrington et al., 2015) in a process which isself-propagating and autocatalytic. Phosphorylation is inhibitory toaggregation (Lai et al., 2016) and is unlikely to drive of the cascade(Mukaetova-Ladinska et al., 2000; Schneider et al., 1999; Wischik etal., 1995). Direct inhibition of tau aggregation represents a plausiblepoint for therapeutic intervention.

Methylthioninium (MT) acts as a tau aggregation inhibitor (TAI) invitro, (Wischik et al., 1996; Harrington et al., 2015) dissolves PHFsfrom Alzheimer's disease brain tissue, (Wischik et al., 1996) andreduces tau pathology and associated behavioural deficits in transgenicmouse tau models at brain concentrations consistent with human oraldosing. (Melis et al., 2015; Baddeley et al., 2015) MT has also beenshown to inhibit other disease-associated protein aggregation (see e.g.WO2007/110629).

MT is a redox molecule and, depending on environmental conditions (e.g.,pH, oxygen, reducing agents), exists in equilibrium between a reduced[leucomethylthioninium (LMT)] and oxidized form (MT⁺).

WO96/30766 describes such MT containing compounds for use in thetreatment and prophylaxis of various diseases, including AD and LewyBody Disease. One example compound was methylthioninium chloride (“MTC”)commonly known as methylene blue, which is the chloride salt of theoxidized form of methylthioninium (MT) i.e. MT⁺.

WO96/30766 describes, in the case of oral administration, a daily dosageof about 50 mg to about 700 mg, preferably about 150 mg to about 300 mg,divided in preferably 1-3 unit doses.

WO2007/110630 discloses certain specific diaminophenothiazine compoundsrelated to MTC, including (so-called) ETC, DEMTC, DMETC, DEETC, MTZ,ETZ, MTI, MTILHI, ETI, ETLHI, MTN, and ETN, which are useful as drugs,for example in the treatment of Alzheimer's disease.

WO2007/110630 describes dosage units comprising 20 to 300 mg of3,7-diaminophenothiazine (DAPTZ) compounds described therein e.g. 30 to200 mg, for example 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. A suitabledose of the DAPTZ compound is suggested in the range of about 100 ng toabout 25 mg (more typically about 1 μg to about 10 mg) per kilogram bodyweight of the subject per day e.g. 100 mg, 3 times daily, 150 mg, 2times daily, 200 mg, 2 times daily. A dosage of 50 mg 3 or 4 times dailyis also discussed.

A preliminary pharmacokinetic model for methylene blue, based on studiesof urinary excretion data sets in humans, dogs and rats, was proposed byDiSanto and Wagner, J Pharm Sci 1972, 61:1086-1090 and 1972,61:1090-1094 and Moody et al., Biol Psych 1989, 26: 847-858.

Peter et al. (2000) Eur J Clin Pharmacol 56: 247-250 provided a modelwhich integrated blood level data, which contradicted the earlier datafrom DiSanto and Wagner as regards terminal elimination half-life.

May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398)showed that human erythrocytes sequentially reduce and take up MTC i.e.that MTC itself is not taken up by the cells but rather that it is thereduced from of MT that crosses the cell membrane. They also showed thatthe rate of uptake is enzyme dependent; and that both oxidised andreduced MT are concentrated in cells (reduced MT re-equilibrates onceinside the cell to form oxidised MT).

Based on these and other disclosures, it is believed that orallyadministered MTC and similar drugs are taken up in the gut and enter thebloodstream, with unabsorbed drug percolates down the alimentary canal,to the distal gut. One important undesired side-effect is the effect ofthe unabsorbed drug in the distal gut, for example, sensitisation of thedistal gut and/or antimicrobial effects of the unabsorbed drug on florain the distal gut, both leading to diarrhoea.

MTC was tested clinically in a phase 2 study (Wischik et al., 2015).Although the minimum safe and effective dose was identified as 138mg/day, a higher dose of 218 mg/day had limited efficacy due toabsorption limitations, most likely due to the need for the MT⁺ to bereduced to the leuco-MT (LMT) form to permit efficient absorption bypassive diffusion.

WO2009/044127 disclosed the results of a phase 2 clinical trial, whichindicated that MTC had two systemic pharmacological actions: cognitiveeffects and haematological effects, but that these actions wereseparable. Specifically the cognitive effects did not show a monotonicdose-response relationship, whereas the haematological effects did. Itwas proposed that two distinct species were responsible for the twotypes of pharmacological activity: MTC absorbed as the unchargedLeuco-MT form being responsible for the beneficial cognitive activity,and MTC absorbed as an oxidised dimeric species being responsible forthe oxidation of haemoglobin. WO2009/044127 described how dosage formscould be used to maximise the bioavailability of the therapeuticallyactive (cognitively effective) species whether dosing with oxidised orleuco-DAPTZ compounds.

Since it is the reduced form of MT that is taken up by cells, it hasbeen proposed to administer a reduced form to patients. This may alsoreduce reliance on the rate limiting step of enzymatic reduction.

MTC, a phenothiazin-5-ium salt, may be considered to be an “oxidizedform” in relation to the corresponding 10H-phenothiazine compound,N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine, which may beconsidered to be a “reduced form”:

The “reduced form” (or “leuco form”) is known to be unstable and can bereadily and rapidly oxidized to give the corresponding “oxidized” form.

WO 02/055720 discloses the use of reduced forms of certaindiaminophenothiazines for the treatment of protein aggregating diseases,primarily tauopathies. Based on in vitro activity for the reduced formsof diaminophenothiazines therein, a suggested daily dosage was 3.2-3.5mg/kg, and dosages of 20 mg t.d.s., 50 mg t.d.s. or 100 mg t.d.s.,combined with 2×mg ratio of ascorbic acid in such a manner as to achievemore than 90% reduction prior to ingestion were also described.

WO2007/110627 disclosed certain 3,7-diamino-10H-phenothiazinium salts,effective as drugs or pro-drugs for the treatment of diseases includingAlzheimer's disease. These compounds are also in the “reduced” or“leuco” form when considered in respect of MTC. Theseleucomethylthioninium compounds were referred to as “LMTX” salts, andincluded the following salts:

WO2012/107706 described other LMTX salts having superior properties tothe LMTX salts listed above, including leuco-methylthioniniumbis(hydromethanesulfonate) (LMTM):

Specifically LMTM retains TAI activity in vitro and in vivo (Wischik etal, 1996; Harrington et al., 2015; Melis et al., 2015) has superiorpharmaceutic properties in terms of solubility and pKa, and is notsubject to the absorption limitations of the MT⁺ form.²⁴ (Baddeley etal., 2015)

WO2007/110627 and WO2012/107706 describes dosage units comprising 20 to300 mg of the DAPTZ compounds described therein e.g. 30 to 200 mg, forexample 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. A suitable dose of theDAPTZ compound is suggested in the range of about 100 ng to about 25 mg(more typically about 1 μg to about 10 mg) per kilogram body weight ofthe subject per day e.g. 100 mg, 3 times daily, 150 mg, 2 times daily,200 mg, 2 times daily.

DISCLOSURE OF THE INVENTION

The present inventors have conducted a 15-month double-blind randomisedcontrolled phase 3 trial in mild to moderate AD patient to test thesafety and efficacy of LMTM.

Doses of LMTM at 75 mg and 125 mg given twice daily (b.i.d.) werecompared with a control dose of 4 mg b.i.d. The control dosage wasdetermined from prior repeat-dose phase 1 studies as the minimum neededto maintain blinding with respect to discolouration of excreta.

Unexpectedly, the 4 mg b.i.d. dose showed therapeutic benefits. Theefficacy profiles were similar in mild and moderate subjects for most ofthe measured outcomes.

The reason for the efficacy of this low dose of MT compound, is unclear.It has previously been shown that the absorption and distribution of MTto the brain is complex, and likely to be mediated via red cells ratherthan plasma (Baddeley et al., 2015) providing a route which protects MTfrom first-pass metabolism. In the same study MT uptake into red cellswas approximately 20-fold higher in vivo when as administeredintravenously as LMTM compared with MTC, most likely due to direct redcell uptake of LMT by passive diffusion without need for prior reductionof MT⁺ as is the case for MTC (Baddeley et al., 2015; May et al., 2004).Without wishing to be bound by theory, the results of the present studysuggest that MT uptake and distribution are capacity-limited by theamount that red cells can take up whilst within the portal circulation.

Unlike an earlier phase 2 study, which was conducted in subjects nottaking AD-labelled co-medications, patients in the phase 3 trial werepermitted to enter whether or not they were not taking thesemedications, as it was considered infeasible for them to be restrictedgiven their extensive use.

Unexpectedly, treatment benefit in AD (according to the trial criteria)was restricted to patients taking LMTM as monotherapy. By contrast, thedecline seen at corresponding doses in patients taking LMTM incombination with AD-labelled treatments (acetylcholinesterase inhibitors[AChEIs] and\or memantine) who were the majority, was indistinguishableon all parameters from that seen in the control arm.

The reason for the loss of benefit on clinical and volumetric outcomesin AD when LMTM is combined with symptomatic AD treatments is unclear,although a possible contributory factor may be induction of themultidrug resistance protein 1 (MDR1), a transporter which isupregulated by AChEIs and memantine. This transporter may directly orindirectly lead to reduction of the levels of MT from the site ofaction.

Irrespective of the mechanism, the disclosure herein indicates that muchlower doses of MT than previously envisaged can produce substantialclinical benefits whilst being well tolerated and having fewer sideeffects than the higher doses.

In respect of AD treatments, such treatments would preferably be amonotherapy, or at least introduced either prior to or followingcessation of the currently available AD treatments AChEIs and memantine,

Although the term “low dose” or “low dosage” has been used in relationto MT-containing compounds in prior art publications, there is noteaching or suggestion in those publications of the utility of thepresent invention.

For example:

Telch, Michael J., et al. “Effects of post-session administration ofmethylene blue on fear extinction and contextual memory in adults withclaustrophobia.” American Journal of Psychiatry 171.10 (2014):1091-1098: this publication refers to the use of “low-dose methyleneblue” on retention of fear extinction and contextual memory followingfear extinction training. The paper reports that “Methylene blue is adiamino phenothiazine drug that at low doses (0.5-4 mg/kg) hasneurometabolic-enhancing properties. The dosages used in the publicationwere 260 mg/day for adult participants, corresponding to a 4 mg/kg dose.

Gonzalez-Lima F and Auchter A (2015) “Protection againstneurodegeneration with low-dose methylene blue and near-infrared light”.Front. Cell. Neurosci. 9:179. doi: 10.3389/fncel.2015.00179: thispublication discusses the cellular mechanisms mediating theneuroprotective effects of low doses of methylene blue and near-infraredlight. It refers to earlier work citing 0.5-4 mg/kg of methylene blue assafe and effective.

Alda, Martin, et al. “Methylene blue treatment for residual symptoms ofbipolar disorder: randomised crossover study.” The British Journal ofPsychiatry (2016): doi: 10.1192/bjp.bp.115.173930: this publicationdescribed the use of a 15 mg “low dose” of methylene blue as a placeboin a 6 month trial. The “active dose” was 195 mg. In each case the dosewas split three times daily.

Rodriguez, Pavel, et al, “Multimodal Randomized Functional MR Imaging ofthe Effects of Methylene Blue in the Human Brain.” Radiology (2016):152893: this publication also refers to the ‘known’ pharmacokinetic andside effects of “low-dose” (0.5-4.0 mg/kg) methylene blue, which arecontrasted with the effects of dosages greater than 10 mg/kg. Thedosages used in the publication were 280 mg/day for adult participants,approximating to a 4 mg/kg dose.

Naylor et al. (1986) “A two-year double-blind crossover trial of theprophylactic effect of methylene blue in manic-depressive psychosis”.Biol. Psychiatry 21:915-920 and Naylor et al. (1987) A controlled trialof methylene blue in severe depressive psychosis. Biol. Psychiatry22:657-659: these studies used 15 mg/day methylene, nominally as aplacebo vs. a treatment of 300 mg/day methylene blue. However in thelatter paper the authors proposed that the placebo dosage may act as anantidepressant.

Thus in one aspect there is disclosed a method of treatment of aneurodegenerative disorders of protein aggregation in a subject,

-   -   which method comprises orally administering to said patient an        methylthioninium (MT) containing compound,    -   wherein said administration provides a total of between 0.5 and        20 mg of MT to the subject per day, optionally split into 2 or        more doses.

The total dose may be from around any of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4mg to around any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 mg.

An example dosage is 1 to 20 mg.

A further example dosage is 2 to 15 mg.

A further example dosage is 3 to 10 mg.

A further preferred dosage is 3.5 to 7 mg.

A further preferred dosage is 4 to 6 mg.

As explained below, when administering MT in the reduced (LMT) form, itmay be desired to use a smaller total amount within the recited range,compared to the oxidised (MT⁺) form.

As explained below, when administering the MT dose split in a largernumber of doses/day it may be desired to use a smaller total amountwithin the recited range, compared to a single daily dosing, or asmaller number of doses per day.

In other embodiments the neurodegenerative disorder may be AD.

As explained herein, in some embodiments the treatment will be amonotherapy, or at least will exclude co-medication with AChEIs andmemantine,

In other embodiments the neurodegenerative disorder may be aneurodegenerative disorder other than AD.

Also provided herein are methods of prophylactic treatment ofneurodegenerative disorders of protein aggregation.

Also provided herein are novel dosage forms containing low unit doses ofMT compounds, for example high purity MT compounds, for example MT⁺compounds or LMTX compounds.

These aspects and embodiments will now be described in more detail:

Methylthioninium Moiety

Structure

IUPAC N3,N3,N7,N7-tetramethyl-10H- N3,N3,N7,N7-phenothiazine-3,7-diamine tetramethylphenothiazin-5-ium- 3,7-diamineComposition Formula Weight: 285.41(1) Formula Weight: 284.40(1) ExactMass: 285.1299683(1) Exact Mass: 284.1215947(1) Formula: C₁₆H₁₉N₃SFormula: C₁₆H₁₈N₃S Composition: C 67.33% H 6.71% Composition: C 67.57% H6.38% N 14.72% S 11.23% N 14.78% S 11.27% Synonym leucomethylthioninium(LMT) oxidized methylthioninium (MT⁺)

The MT⁺-containing compounds used in the present invention can containMT in either reduced or oxidised form. The “MT” is the activeingredient, which is to say that it is present to provide the recitedtherapeutic effect. Specifically, the compounds may comprise either ofthe MT moieties described above. The MT moieties per se described aboveare not stable. They will therefore be administered as MT compounds—forexample LMT or MT⁺ salts.

MT⁺ salts will generally include one or more anionic counter ions (X⁻)to achieve electrical neutrality. The compounds may be hydrates,solvates, or mixed salts of the MT⁺ salt.

LMT containing compounds will generally be stabilised, for example bythe presence of one or more protic acids e.g. two protic acids.

The MT content of such salts can be readily calculated by those skilledin the art based on the molecular weight of the compound, and themolecular weight of the MT moiety. Examples of such calculations aregiven herein.

LMT Compounds

Preferably the MT compound is an LMT compound.

Preferably the MT compound is an “LMTX” compound of the type describedin WO2007/110627 or WO2012/107706.

Thus the compound may be selected from compounds of the followingformula, or hydrates or solvates thereof:

Each of H_(n)A and H_(n)B (where present) are protic acids which may bethe same or different.

By “protic acid” is meant a proton (H⁺) donor in aqueous solution.Within the protic acid A⁻ or B⁻ is therefore a conjugate base. Proticacids therefore have a pH of less than 7 in water (that is theconcentration of hydronium ions is greater than 10⁻⁷ moles per litre).

In one embodiment the salt is a mixed salt that has the followingformula, where HA and HB are different mono-protic acids:

However preferably the salt is not a mixed salt, and has the followingformula:

wherein each of H_(n)X is a protic acid, such as a di-protic acid ormono-protic acid.

In one embodiment the salt has the following formula, where H₂A is adi-protic acid:

Preferably the salt has the following formula which is a bis monoproticacid:

Examples of protic acids which may be present in the LMTX compounds usedherein include:

Inorganic acids: hydrohalide acids (e.g., HCl, HBr), nitric acid (HNO₃),sulphuric acid (H₂SO₄)

Organic acids: carbonic acid (H₂CO₃), acetic acid (CH₃COOH),methanesulfonic acid, 1,2-Ethanedisulfonic acid, ethansulfonic acid,Naphthalenedisulfonic acid, p-toluenesulfonic acid,

Preferred acids are monoprotic acid, and the salt is a bis(monoproticacid) salt.

A preferred MT compound is LMTM:

1

LMT•2MsOH (LMTM) 477.6 (1.67)

The anhydrous salt has a molecular weight of around 477.6. Based on amolecular weight of 285.1 for the LMT core, the weight factor for usingthis MT compound in the invention is 1.67. By “weight factor” is meantthe relative weight of the pure MT containing compound vs. the weight ofMT which it contains.

Other weight factors can be calculated for example MT compounds herein,and the corresponding dosage ranges can be calculated therefrom.

Therefore the invention embraces a total daily dose of around 0.8 to 33mg/day of LMTM.

More preferably around 6 to 12 mg/day of LMTM total dose is utilised,which corresponds to about 3.5 to 7 mg MT.

Other example LMTX compounds are as follows. Their molecular weight(anhydrous) and weight factor is also shown:

2

LMT•2EsOH 505.7 (1.77) 3

LMT•2TsOH 629.9 (2.20) 4

LMT•2BSA 601.8 (2.11) 5

LMT•EDSA 475.6 (1.66) 6

LMT•PDSA 489.6 (1.72) 7

LMT•NDSA 573.7 (2.01) 8

LMT•2HCl 358.33 (1.25)

The dosages described herein with respect to MT thus apply mutatismutandis for these MT containing compounds, as adjusted for theirmolecular weight.

Oxidised MT Compounds

In another embodiment the MT compound is an MT⁺ compound.

Preferably the MT compound is an MT⁺ compound of the type described inWO96/30766 or WO2007/110630.

Thus the compound may be selected from compounds of the followingformula, or hydrates, solvates, or mixed salts thereof:

Where X⁻ is an anionic counter ion.

In some embodiments of the present invention the MT⁺ compound is MTC,for example a “high purity” MTC as described below.

In some embodiments of the present invention the MT⁺ compound is notMTC.

9

MTC methyl-thioninium chloride

As explained in WO2011/036561 and WO2011/036558, MTC occurs in a numberof polymorphic forms having different levels of hydration.

In some embodiments of the present invention, the MT⁺ compound is a highpurity MTC. In this context ‘high purity’ is defined by one or more ofthe criteria set out below.

In some embodiments, the MTC has a purity of greater than 97%.

In some embodiments, the MTC has a purity of greater than 98%.

In some embodiments, the MTC has a purity of greater than 99%.

In some embodiments, the MTC has less than 2% Azure B as impurity.

In some embodiments, the MTC has less than 1% Azure B as impurity.

In some embodiments, the MTC has less than 0.5% Azure B as impurity.

In some embodiments, the MTC has less than 0.1% Azure B as impurity.

In some embodiments, the MTC has less than 0.15% Azure A as impurity.

In some embodiments, the MTC has less than 0.10% Azure A as impurity.

In some embodiments, the MTC has less than 0.05% Azure A as impurity.

In some embodiments, the MTC has less than 0.15% Azure C as impurity.

In some embodiments, the MTC has less than 0.10% Azure C as impurity.

In some embodiments, the MTC has less than 0.05% Azure C as impurity.

In some embodiments, the MTC has less than 0.13% MVB (Methylene VioletBernstein) as impurity.

In some embodiments, the MTC has less than 0.05% MVB as impurity.

In some embodiments, the MTC has less than 0.02% MVB as impurity.

All percentage purities recited herein are by weight unless otherwisespecified.

In some embodiments, the MTC has an elementals purity that is betterthan that specified by the European Pharmacopeia (EP).

As used herein, the term ‘elementals purity’ pertains to the amounts ofthe twelve (12) metals specified by the European Pharmacopeia: Al, Cd,Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb, and Zn. The current edition of theEuropean Pharmacopeia (8^(th) Edition, supplementum 8.8) specifies thefollowing limits for these metals:

European Pharmacopeia 8.8 (EP8.8) Element Maximum content (μg/g)Aluminium (Al) 100 Cadmium (Cd) 1 Chromium (Cr) 100 Copper (Cu) 300 Tin(Sn) 10 Iron (Fe) 200 Manganese (Mn) 10 Mercury (Hg) 1 Molybdenum (Mo)10 Nickel (Ni) 10 Lead (Pb) 10 Zinc (Zn) 100

In one embodiment, the MTC has an elementals purity (e.g. for each ofAl, Cd, Cr, Cu, Sn, Fe, Mn, Hg, Mo, Ni, Pb, and Zn) which is equal to orbetter than (i.e. lower than) the EP8.8 values set out in the tableabove.

In one embodiment, the MTC has an elementals purity which is equal to orbetter than 0.9 times the EP8.8 values set out in the table above.

In one embodiment, the MTC has an elementals purity which is equal to orbetter than 0.8 times the EP8.8 values set out in the table above.

In one embodiment, the MTC has an elementals purity which is equal to orbetter than 0.7 times the EP8.8 values set out in the table above.

In one embodiment, the MTC has an elementals purity which is equal to orbetter than 0.5 times the EP8.8 values set out in the table above.

(For example, 0.5 times the EP8.8 values as set out above are 50 μg/gAl, 0.5 μg/g Cd, 50 μg/g Cr, etc.)

In one embodiment the MTC has a chromium level that is equal to orbetter than (i.e. lower than) 100 μg/g.

In one embodiment the MTC has a chromium level that is equal to orbetter than (i.e. lower than) 10 μg/g.

In one embodiment the MTC has a copper level that is equal to or betterthan (i.e. lower than) 300 μg/g.

In one embodiment the MTC has a copper level that is equal to or betterthan (i.e. lower than) 100 μg/g.

In one embodiment the MTC has a copper level that is equal to or betterthan (i.e. lower than) 10 μg/g.

In one embodiment the MTC has an iron level that is equal to or betterthan (i.e. lower than) 200 μg/g.

In one embodiment the MTC has an iron level that is equal to or betterthan (i.e. lower than) 100 μg/g.

All plausible and compatible combinations of the above purity grades aredisclosed herein as if each individual combination was specifically andexplicitly recited.

In particular embodiments, the MTC is a high purity MTC wherein ‘highpurity’ is characterised by a purity of greater than 98% and one or moreof the following:

(i) less than 2% Azure B as impurity;

(ii) less than 0.13% MVB (Methylene Violet Bernstein) as impurity; or

(iii) an elementals purity better than the European Pharmacopeia limitsof less than 100 μg/g Aluminium (Al); less than 1 μg/g Cadmium (Cd);less than 100 μg/g Chromium (Cr); less than 300 μg/g Copper (Cu); lessthan 10 μg/g Tin (Sn); less than 200 μg/g Iron (Fe); less than 10 μg/gManganese (Mn); less than 1 μg/g Mercury (Hg); less than 10 μg/gMolybdenum (Mo); less than 10 μg/g Nickel (Ni); less than 10 μg/g Lead(Pb); and less than 100 μg/g Zinc (Zn).

In particular embodiments, the MTC is a high purity MTC whereinhigh-purity is characterised by a purity of greater than 98% and one ormore of the following:

(i) less than 1% Azure B as impurity;

(ii) less than 0.15% Azure A as impurity;

(iii) less than 0.15% Azure C as impurity;

(iv) less than 0.13% Methylene Violet Bernthsen (MVB) as impurity;

(v) an elementals purity better than the European Pharmacopeia limits ofless than 100 μg/g Aluminium (Al); less than 1 μg/g Cadmium (Cd); lessthan 100 μg/g Chromium (Cr); less than 300 μg/g Copper (Cu); less than10 μg/g Tin (Sn); less than 200 μg/g Iron (Fe); less than 10 μg/gManganese (Mn); less than 1 μg/g Mercury (Hg); less than 10 μg/gMolybdenum (Mo); less than 10 μg/g Nickel (Ni); less than 10 μg/g Lead(Pb); and less than 100 μg/g Zinc (Zn).

In particular embodiments, the MTC is a high purity MTC whereinhigh-purity is characterised by a purity of greater than 98% and one ormore of the following:

(i) less than 1% Azure B as impurity;

(ii) less than 0.15% Azure A as impurity;

(iii) less than 0.15% Azure C as impurity;

(iv) less than 0.05% Methylene Violet Bernthsen (MVB) as impurity; or

(v) an elementals purity better than the European Pharmacopeia limits ofless than 100 μg/g Aluminium (Al); less than 1 μg/g Cadmium (Cd); lessthan 100 μg/g Chromium (Cr); less than 300 μg/g Copper (Cu); less than10 μg/g Tin (Sn); less than 200 μg/g Iron (Fe); less than 10 μg/gManganese (Mn); less than 1 μg/g Mercury (Hg); less than 10 μg/gMolybdenum (Mo); less than 10 μg/g Nickel (Ni); less than 10 μg/g Lead(Pb); and less than 100 μg/g Zinc (Zn).

In particular embodiments, the MTC is a high purity MTC whereinhigh-purity is characterised by at least 98% purity and less than 1%Azure B as impurity.

In particular embodiments, the MTC is a high purity MTC whereinhigh-purity is characterised by:

(i) at least 98% purity

(i) less than 1% Azure B as impurity; and

(ii) an elementals purity better than the European Pharmacopeia limitsof less than 100 μg/g Aluminium (Al); less than 1 μg/g Cadmium (Cd);less than 100 μg/g Chromium (Cr); less than 300 μg/g Copper (Cu); lessthan 10 μg/g Tin (Sn); less than 200 μg/g Iron (Fe); less than 10 μg/gManganese (Mn); less than 1 μg/g Mercury (Hg); less than 10 μg/gMolybdenum (Mo); less than 10 μg/g Nickel (Ni); less than 10 μg/g Lead(Pb); and less than 100 μg/g Zinc (Zn).

In particular embodiments, the MTC is a high purity MTC whereinhigh-purity is characterised by at least 98% purity and an elementalspurity better than the European Pharmacopeia limits of less than 100μg/g Aluminium (Al); less than 1 μg/g Cadmium (Cd); less than 100 μg/gChromium (Cr); less than 300 μg/g Copper (Cu); less than 10 μg/g Tin(Sn); less than 200 μg/g Iron (Fe); less than 10 μg/g Manganese (Mn);less than 1 μg/g Mercury (Hg); less than 10 μg/g Molybdenum (Mo); lessthan 10 μg/g Nickel (Ni); less than 10 μg/g Lead (Pb); and less than 100μg/g Zinc (Zn).

Methods for the production of ‘high purity’ diaminophenothiaziniumcompounds, including MTC, are described, for example, in WO2006/032879and WO2008/007074 (WisTa Laboratories Ltd) and in WO2008/006979(Provence Technologies).

A preferred MTC polymorph for use in the methods and compositionsdescribed herein is ‘form A’ described in WO2011/036561 which is apentahydrate, at a “high purity” described above. That has a molecularweight of around 409.9. Based on a molecular weight of 284.1 for the MT⁺core, the weight factor for using this MT compound in the invention is1.44.

Other weight factors can be calculated for example MT compounds herein,and the corresponding dosage ranges can be calculated therefrom.

Therefore the invention embraces a total daily dose of around 0.7 to 29mg/day of MTC·5H₂O.

More preferably around 5 to 10 mg/day of MTC·5H₂O total dose isutilised, which corresponds to about 3.5 to 7 mg MT.

Other example MT compounds are described in WO2007/110630. Theirmolecular weight (anhydrous) and weight factor is also shown:

MT⁺ anhydrous Compound Molecular weight weight factor 10 MTC•0.5ZnCl₂388.0 1.36 11 MTI 411.3 1.45 12 MTI•HI 539.2 2.73 13 MT•NO₃ 346.4 1.22

The dosages described herein with respect to MT thus apply mutatismutandis for these MT containing compounds, as adjusted for theirmolecular weight, and for choice of hydrate if used. For exampleMTC·0.5ZnCl₂ (also referred to as ‘METHYLENE BLUE ZINC CHLORIDE DOUBLESALT; CI 52015) may be obtained commercially as a monohydrate by severalsuppliers, which would have a molecular weight higher by 18, andcorrespondingly altered weight factor. MTI is reportedly available as ahemihydrate,

Adsorption Factors

As explained herein, the present inventors have determined that,unexpectedly, low doses of MT salts showed therapeutic benefits in aneurodegenerative disorder of protein aggregation. This was demonstratedusing an example LMTX salt. This finding has implications for the dosingof both LMT and MT⁺ salts.

The present inventors have determined that dosing with LMTX saltspermits more efficient adsorption, compared with MT⁺ salts. Typically MTadsorption may be around 1.5× greater when delivered as an LMTX salt asopposed to an MT⁺ salts. This 1.5 factor may be termed herein an“adsorption factor”.

Therefore in certain embodiments of the invention, the dosed amount ofMT⁺ salt may be higher than when using LMTX salt to achieve a similarplasma concentration.

Thus one preferred dosage of MT⁺ salt may be about 5.25 to 10.5 mg MT,which is expected to provide a similar adsorbed dosage as 3.5 to 7 mg MTwhen delivered as LMTX.

Any of the MT compounds described herein, may be formulated with areducing agent. In particular, MT⁺ salts such as MTC may be formulatedwith a reducing agent such as ascorbate, and then lyophilized (asdescribed in WO02/055720). This is expected to improve adsorption of theMT delivered by the compound.

In the various aspects of the invention described herein (as they relateto an MT-containing compound) this may optionally be any of thosecompounds described above:

In one embodiment, it is compound 1.

In one embodiment, it is compound 2.

In one embodiment, it is compound 3.

In one embodiment, it is compound 4.

In one embodiment, it is compound 5.

In one embodiment, it is compound 6.

In one embodiment, it is compound 7.

In one embodiment, it is compound 8.

In one embodiment, it is compound 9.

In one embodiment, it is compound 10.

In one embodiment, it is compound 11.

In one embodiment, it is compound 12.

In one embodiment, it is compound 13.

Or the compounds may be a hydrate, solvate, or mixed salt of any ofthese.

Accumulation Factors

As will be appreciated by those skilled in the art, for a given dailydosage, more frequent dosing will lead to greater accumulation of adrug.

The present inventors have derived estimated accumulation factors for MTas follows:

Observed plasma Relative Dosing accumulation for MT accumulation Oncedaily 1.29^(extrapolated) 1 Twice daily 1.47 1.13 Three-times daily 1.651.28

For example, considering a total daily dose of 3.5 to 7 mg MT:

When given as a single daily dose, this may equate to an accumulation ofMT in plasma of 4.5 to 8

When split b.i.d., this may equate to an accumulation of MT in plasma of5.1 to 10.3

When split t.i.d., this may equate to an accumulation of MT in plasma of5.8 to 11.6

Therefore in certain embodiments of the invention, the total daily dosedamount of MT compound may be lower, when dosing more frequently (e.g.twice a day [bid] or three times a day [tid]).

In one embodiment, LMTM is administered around 9 mg/once per day; 4 mgb.i.d.; 2.3 mg t.i.d (based on weight of LMTM)

In one embodiment, MTC:5H2O is administered around 10.6 mg/once per day;6 mg b.i.d.; 2.8 mg t.i.d (based on weight of MTC:5H2O).

Treatment and Prophylaxis

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound of the invention, or a material, compositionor dosage from comprising said compound, which is effective forproducing some desired therapeutic effect, commensurate with areasonable benefit/risk ratio, when administered in accordance with adesired treatment regimen. The present inventors have demonstrated thata therapeutically-effective amount of an MT compound in respect of thediseases of the invention can be much lower than was hitherto understoodin the art.

The invention also embraces treatment as a prophylactic measure is alsoincluded.

Thus the invention also provides

-   -   a method of prophylaxis of a neurodegenerative disorders of        protein aggregation    -   in a subject,        -   which method comprises orally administering to said patient            an MT containing compound,        -   wherein said administration provides a total of between 0.5            and 20 mg of MT to the subject per day, optionally split            into 2 or more doses.

The term “prophylactically effective amount,” as used herein, pertainsto that amount of a compound of the invention, or a material,composition or dosage from comprising said compound, which is effectivefor producing some desired prophylactic effect, commensurate with areasonable benefit/risk ratio, when administered in accordance with adesired treatment regimen.

“Prophylaxis” in the context of the present specification should not beunderstood to circumscribe complete success i.e. complete protection orcomplete prevention. Rather prophylaxis in the present context refers toa measure which is administered in advance of detection of a symptomaticcondition with the aim of preserving health by helping to delay,mitigate or avoid that particular condition.

Combination Treatments and Monotherapy

The term “treatment” includes “combination” treatments and therapies, inwhich two or more treatments or therapies for the same neurodegenerativedisorder of protein aggregation, are combined, for example, sequentiallyor simultaneously. These may be symptomatic or disease modifyingtreatments.

The particular combination would be at the discretion of the physician.

In combination treatments, the agents (i.e., an MT compound as describedherein, plus one or more other agents) may be administeredsimultaneously or sequentially, and may be administered in individuallyvarying dose schedules and via different routes. For example, whenadministered sequentially, the agents can be administered at closelyspaced intervals (e.g., over a period of 5-10 minutes) or at longerintervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periodsapart where required), the precise dosage regimen being commensuratewith the properties of the therapeutic agent(s).

An example of a combination treatment of the invention would be an agentwhich is MT-containing compound at the specified dosage in combinationwith an agent which is an inhibitor of amyloid precursor protein tobeta-amyloid (e.g., an inhibitor of amyloid precursor protein processingthat leads to enhanced generation of beta-amyloid).

In other embodiments the treatment is a “monotherapy”, which is to saythat the MT-containing compound is not used in combination (within themeaning discussed above) with another active agent for treating the sameneurodegenerative disorder of protein aggregation in the subject.

As explained below, in the present invention, when treating AD at least,it is preferred that the treatment does not include administration ofeither or both of: an acetylcholinesterase inhibitor or anN-methyl-D-aspartate receptor antagonist. The MT-compound basedtreatment of AD may optionally be a monotherapy.

Duration of Treatment

For treatment of the neurodegenerative disorder of protein aggregationdescribed herein, a treatment regimen based on the low dose MT compoundswill preferably extend over a sustained period of time. The particularduration would be at the discretion of the physician.

For example, the duration of treatment may be:

At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer.

At least 2, 3, 4, 5 years, or longer.

Between 6 and 12 months.

Between 1 and 5 years.

Where the disorder is AD, duration may be such as to achieve any one ormore of:

A 3, 4 or 5-point improvement on the 11-item Alzheimer's DiseaseAssessment Scale—cognitive subscale (ADAS-cog) over a 52-week period;

4, 5 or 6 point improvement on the 23-item Alzheimer's DiseaseCooperative Study Activities of Daily Living (ADCS-ADL) over a 52-weekperiod;

A reduction in the increase of Lateral Ventricular Volume (LVV), asmeasured by the Ventricular Boundary Shift Integral (VBSI) of 1 or 2 cm³over a 52-week period.

For prophylaxis, the treatment may be ongoing.

In all cases the treatment duration will generally be subject to adviceand review of the physician.

Oral Dosage Forms

The MT compound of the invention, or pharmaceutical compositioncomprising it, is administered to a subject/patient orally.

Pharmaceutical Dosage Forms

Another aspect of the invention therefore provides a compositioncomprising a compound as described herein, and a pharmaceuticallyacceptable carrier or diluent.

In some embodiments, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a compound asdescribed herein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

In some embodiments, the composition is a pharmaceutical compositioncomprising at least one compound, as described herein, together with oneor more other pharmaceutically acceptable ingredients well known tothose skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In some embodiments, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising an MT compound as describedherein (e.g., obtained by, or obtainable by, a method as describedherein; having a purity as described herein; etc.), and apharmaceutically acceptable carrier, diluent, or excipient.

The “MT compound”, although present in relatively low amount, is theactive agent of the dosage unit, which is to say is intended to have thetherapeutic or prophylactic effect in respect of a neurodegenerativedisorder of protein aggregation. Rather, the other ingredients in thedosage unit will be therapeutically inactive e.g. carriers, diluents, orexcipients. Thus, preferably, there will be no other active ingredientin the dosage unit, no other agent intended to have a therapeutic orprophylactic effect in respect of a disorder for which the dosage unitis intended to be used.

In some embodiments, the dosage unit is a tablet.

In some embodiments, the dosage unit is a capsule.

In some embodiments, said capsules are gelatine capsules.

In some embodiments, said capsules are HPMC(hydroxypropylmethylcellulose) capsules.

In some embodiments, the amount of MT in the unit is 0.5 to 10 mg.

An example dosage unit may contain 1 to 10 mg of MT.

A further example dosage unit may contain 2 to 9 mg of MT.

A further example dosage unit may contain 3 to 8 mg of MT.

A further preferred dosage unit may contain 3.5 to 7 mg of MT.

A further preferred dosage unit may contain 4 to 6 mg of MT.

In some embodiments, the amount is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 5,6, 7, 8, 9, 10 mg of MT.

Using the weight factors described or explained herein, one skilled inthe art can select appropriate amounts of an MT containing compound touse in oral formulations.

As explained above, the MT weight factor for LMTM is 1.67. Since it isconvenient to use unitary or simple fractional amounts of activeingredients, non-limiting example LMTM dosage units may include 1.5, 2,2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18 mg etc.

As explained above, the MT weight factor for MTC·5H₂O is 1.44. Since itis convenient to use unitary or simple fractional amounts of activeingredients, non-limiting example MTC·5H₂O dosage units may include 1.5,2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20 mg etc.

Nutraceutical Compositions

The “nutraceutical compositions” of the invention comprise a low dose ofMT compound, as described herein, in combination with one or morenutrients in an edible form (for example an oral dosage form).

The novel nutraceutical compositions of the invention can find use assupplements to food and beverages, and as pharmaceutical compositions.

“Nutrients” as used herein refers to the components of nutraceuticalcompositions that serve a biochemical and/or physiological role in thehuman or animal body. “Nutrients” includes such substances as vitamins,minerals, trace elements, micronutrients, antioxidants and the like, aswell as other bioactive materials, such as enzymes, or compoundsbiosynthetically produced by human or animal enzymes; as well as herbsand herbal extracts; fatty acids, amino acids and derivatives thereof.

“Edible form” denotes a composition that can be ingested directly orconverted to an ingestible form, such as, by dissolving in water.

Alternatively, the nutraceutical composition can be in the form of afood or drink, such as a defined portion of a foodstuff (which termincludes both food or drink) supplemented with the defined dosage of MTcompound. These foodstuffs will typically comprise one or more of a fat,a protein, or a carbohydrate.

The term “nutraceutical’ as used herein denotes a usefulness in both thenutritional and pharmaceutical field of application, and the disclosureherein relating to pharmaceutical dosage forms applies mutatis mutandisto the nutraceutical compositions.

Oral dosage forms particularly suitable for nutraceutical compositionsare well known in the art and described in more detail elsewhere herein.They include powders, capsules, pills, tablets, caplets, gelcaps, anddefined portions of edible food items. Liquid forms include solutions orsuspensions. General examples of dosage forms and nutraceutical formsare given, for example in WO2010/078659.

Some examples of nutrients useful in the compositions of the presentinvention are as follows. Any combination of these nutrients isenvisaged by the present invention:

Vitamins

It is reported that B-vitamin supplementation (folic acid [folate,vitamin B₉], vitamin B₁₂, vitamin B₆) can slow the atrophy of specificbrain regions that are a key component of the AD process and that areassociated with cognitive decline. This is particularly the case forelderly subjects with high homocysteine levels (Douaud, Gwenaëlle, etal. “Preventing Alzheimer's disease-related gray matter atrophy byB-vitamin treatment.” Proceedings of the National Academy of Sciences110.23 (2013): 9523-9528; see also Quadri, Pierluigi, et al.“Homocysteine, folate, and vitamin B₁₂ in mild cognitive impairment,Alzheimer disease, and vascular dementia.” The American journal ofclinical nutrition 80.1 (2004): 114-122; Rosenberg I H, Miller J W.Nutritional factors in physical and cognitive functions of elderlypeople. The American journal of clinical nutrition. 1992 Jun. 1;55(6):12375-1243S).

It has been suggested that, along with other antioxidants (see below),vitamin C may have utility in protecting neural tissue, as well aspotentially decreasing β-amyloid generation and acetylcholinesteraseactivity and prevents endothelial dysfunction by regulating nitric oxide(see e.g. Heo J H, Hyon-Lee, Lee K M. The possible role of antioxidantvitamin C in Alzheimer's disease treatment and prevention. AmericanJournal of Alzheimer's Disease & Other Dementias. 2013 March;28(2):120-5).

It has also been suggested that Vitamin E supplementation may have arole to play in AD treatment (see e.g. Mangialasche, Francesca, et al.“Serum levels of vitamin E forms and risk of cognitive impairment in aFinnish cohort of older adults.” Experimental gerontology 48.12 (2013):1428-1435).

Micronutrients, Antioxidants

Micronutrients or antioxidants, such as polyphenols, have been reportedto have benefits in relation to protection or treatment of age-relateddiseases including neurodegenerative ones, particularly cognitiveimpairment and AD.

Micronutrients and\or antioxidants which may be utilised in thenutraceutical compositions described herein include the flavonoids shownin the Table below (reproduced from Mecocci, Patrizia, et al.“Nutraceuticals in cognitive impairment and Alzheimer's disease.”Frontiers in pharmacology 5:147 (2014)):

Flavonoid chemical subgroups and relative food sources:

Groups Molecules Food source FLAVANOLS Catechin, epicatechin, Cocoa andchocolate, epigallocathechin, green tea, grapes epigallocatechin gallate(EGCG) FLAVONOLS Kaempferol, quercetin Onions, apples, green tea,capers, leeks, broccoli FLAVONES Luteolin, apigenin Celery, parsley,rosemary ISOFLAVONES Daidzein, genistein Soy FLAVANONES Hesperetin,naringenin Citrus fruit, tomatoes ANTHOCYANIDINS Pelargonidin, Berryfruits, red wine cyanidine, malvidin

Other micronutrients having potential utility in relation to protectionor treatment of age-related diseases, and described by Mecocci et alinclude:

-   -   Non-flavonoid polyphenols: resveratrol and curcumin,    -   Carotenoids: lycopene, lutein, zeaxanthin, β-cryptoxanthin,        α-carotene, and the most prominent carotenoid, β-carotene,    -   Crocin (the main chemical compound identified in saffron),    -   Diterpenes: for example carnosic and rosmarinic acids are two of        the most important antioxidant compounds in rosemary.

Herbs and Plant Extracts

In addition to the plants described or cross-referenced above inrelation to micronutrients and antioxidants, other plant extracts andherbs are reported to have benefit in CNS disorders—see Kumar, Vikas.“Potential medicinal plants for CNS disorders: an overview.”Phytotherapy Research 20.12 (2006): 1023-1035. These include Ginkgobiloba, Hypericum perforatum (St John's wort), Piper methysticum Forst.(Family Piperaceae) also called kava kava, Valeriana officinalis L.(Valerian), Bacopa monniera (which in India is locally known as Brahmior Jalanimba), Convolvulus pluricaulis (also known as Shankhpushpi orshankapushpi)

Oils and Fats

It is reported that ω-3 polyunsaturated fatty acid (PUFA), for example,may be a promising tool for preventing age-related brain deterioration.Sources of PUFA such as (docosahexaenoic acid (DHA, 22:6) andeicosapentenoic acid (EPA, 20:5) include fish oils (Denis, I., et al.“Omega-3 fatty acids and brain resistance to ageing and stress: body ofevidence and possible mechanisms.” Ageing research reviews 12.2 (2013):579-594.)

Immediate Release Dosage Units

The formulations and compositions (especially pharmaceuticalcompositions) may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

An immediate release product allows the ingredient or active moiety todissolve in the gastrointestinal tract, without causing any delay orprolongation of the dissolution or absorption of the drug. Requirementsfor dissolution testing of immediate release products are set out in theGuidance for Industry (CDER 1997) “Dissolution testing for immediaterelease solid oral dosage forms”, (CDER 1997) “Immediate release solidoral dosage forms—Scale up and Post approval Changes”, ICH Guidance Q6A,Specifications: Test Procedures and Acceptance Criteria For New DrugSubstances And New Drug Products. The most commonly employed dissolutiontest methods as described in the USP and European Pharmacopeia (6thedition) are the basket method (USP 1) and the paddle method (USP 2).The described methods are simple, robust, well standardized, and usedworldwide. They are flexible enough to allow dissolution testing for avariety of drug products. The following parameters influencing thedissolution behaviour may for example be relevant for selecting theappropriate in vitro dissolution test conditions for an immediaterelease solid oral product: apparatus, stirring speed, dissolutionmedium and temperature. Because of the biopharmaceutical properties ofMTC and its expected desirable absorption characteristics in the uppergastrointestinal tract, it was preferable to produce rapidly dissolvingtablets of MTC.

Compositions according to the invention can be dissolution tested in aUSP-2 apparatus in 900 ml of 0.1N HCl, with paddles rotating at 50-75rpm. Compositions according to the invention exhibit at least theacceptance criteria cited for Stage 1 (S1) testing in the USP 32 (TheUnited States Pharmacopeia, edited by the United States PharmacopeialConvention, Inc., 12601 Twinbrook Parkway, Rockville, Md. 20852;Published by Rand McNally, Inc., 32nd Edition, 2008):

Acceptance Criteria: Each tablet achieved 85% dissolution of MTC within30 minutes after insertion of the coated tablet into the 0.1N HCl.

Thus in some embodiments, the MTC based formulations of the invention,when evaluated using this method, provide at least:

75% dissolution of MTC within 45 minutes after insertion of the coatedtablet into the 0.1N HCl; or

85% dissolution of MTC within 30 minutes after insertion of the coatedtablet into the 0.1N HCl;

85% dissolution of MTC within 15 minutes after insertion of the coatedtablet into the 0.1N HCl.

Another aspect of the present invention pertains to methods of making alow dosage MT compound pharmaceutical composition comprising admixing atleast one MT compound, as defined herein, together with one or moreother pharmaceutically acceptable ingredients well known to thoseskilled in the art, e.g., carriers, diluents, excipients, etc. Ifformulated as discrete units (e.g., tablets, etc.), each unit contains apredetermined amount (dosage) of the compound.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

In some embodiments, the pharmaceutically acceptable carrier, diluent,or excipient is or comprises one or both of a glyceride (e.g., Gelucire44/14®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicondioxide (e.g., 2% Aerosil 200®; Colliodal Silicon Dioxide PhEur, USP).

Preferably the pharmaceutical compositions comprising a compound of theinvention, in solid dosage form. The composition preferably furthercomprises at least one diluent suitable for dry compression. Thepharmaceutical composition is characterised in that the compound existsin a substantially stable form.

The pharmaceutical composition will generally also include a lubricant.Examples of lubricants include magnesium stearate, calcium stearate,sodium stearyl fumarate, stearic acid, glycerylbehaptate, polyethyleneglycol, ethylene oxide polymers (for example, those available under theregistered trademark Carbowax from Union Carbide, Inc., Danbury, Conn.),sodium lauryl sulphate, magnesium lauryl stearate, mixtures of magnesiumstearate with sodium lauryl sulphate, and hydrogenated vegetable oil.Preferred lubricants include calcium stearate, magnesium stearate andsodium stearyl fumarate. Most preferred as the lubricant is magnesiumstearate. Lubricants generally comprise from about 0.5 to about 5.0% ofthe total (uncoated) tablet weight. The amount of lubricant employed isgenerally from about 1.0 to about 2.0%, preferably 0.5 to 2.0% w/w.

In addition to the diluent(s) and lubricant(s), other conventionalexcipients may also be present in the pharmaceutical compositions of theinvention. Such additional excipients include disintegrants, binders,flavouring agents, colours and glidants. Some excipients can servemultiple functions, for example as both binder and tablet disintegrant.

A tablet disintegrant may be present in an amount necessary to achieverapid dissolution. Disintegrants are excipients which oppose thephysical forces of particle bonding in a tablet or capsule when thedosage form is placed in an aqueous environment. Examples ofdisintegrants include crosslinked polyvinylpyrrolidone (crospovidone),sodium starch glycolate, crosslinked sodium carboxymethyl cellulose(sodium croscarmellose), and pregelatinized starch. Generally the amountof disintegrant can be from 0 to about 25% w/w, more commonly from about1% to about 15% w/w, and usually less than 10% or less than 5% w/w, ofthe composition.

Binders are excipients which contribute to particle adhesion in a solidformulation. Examples of binders include cellulose derivatives(carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose, microcrystallinecellulose) and sugars such as lactose, sucrose, dextrose, glucose,maltodextrin, and mannitol, xylitol, polymethacrylates,polyvinylpyrrolidone, sorbitol, pregelatinized starch, alginic acids,and salts thereof such as sodium alginate, magnesium aluminum silicate,polyethylene glycol, carrageenan and the like. Generally, the amount ofbinder can vary widely, eg from 0% to 95% w/w of the composition. Asnoted above, excipients may serve multiple functions. For instance, thetabletting diluent may also serve as a binder.

Glidants are substances added to a powder to improve its flowability.Examples of glidants include magnesium stearate, colloidal silicondioxide (such as the grades sold as Aerosil), starch and talc. Glidantsmay be present in the pharmaceutical composition at a level of from 0 toabout 5% w/w. Again, however, it should be noted that excipients mayserve multiple functions. The lubricant, for example magnesium stearate,may also function as a glidant.

Examples of colours that may be incorporated into the pharmaceuticalcompositions of the invention include titanium dioxide and/or dyessuitable for food such as those known as FD&C dyes and natural colouringagents. A colouring agent is unlikely to be used in the powder mixturethat is compressed in accordance with the aspects of the inventiondiscussed above, but may form part of a coating applied to thecomposition, as described below, in which case the colouring agent maybe present in the film coat in an amount up to about 2.0% w/w.

The tablet is desirably coated with a conventional film coating whichimparts toughness, ease of swallowing, and an elegant appearance to thefinal product. Many polymeric film-coating materials are known in theart. A preferred film-coating material is hydroxypropylmethylcellulose(HPMC) or polyvinyl alcohol-part hydrolysed (PVA). HPMC and PVA may beobtained commercially, for example from Colorcon, in coatingformulations containing excipients which serve as coating aids, underthe registered trademark Opadry. Opadry formulations may also containtalc, polydextrose, triacetin, polyethyleneglycol, polysorbate 80,titanium dioxide, and one or more dyes or lakes. Other suitablefilm-forming polymers may also be used, includinghydroxypropylcellulose, vinyl copolymers such as polyvinyl pyrollidoneand polyvinyl acetate, and acrylate-methacrylate copolymers. Use of afilm coating is beneficial for ease of handling and because a bluecoloured uncoated core may stain the inside of the mouth duringswallowing. Coating also improves light stability of the dosage form.

Coating of the tablets may conveniently be carried out using aconventional coating pan. In preferred embodiments of the process, thecoating pan is pre-heated using heated inlet air until the exhausttemperature reaches 35°-55° C., more preferably 40-50° C. This maytypically require application of heated inlet air at an inlettemperature of 45-75° C., preferably 50-65° C., for 10-15 minutes. Thetablet cores containing the active ingredient (e.g. LMTM) are then addedto the coating pan and the aqueous film coat applied. The spray rate iscontrolled such that the bed temperature is maintained at 38-48° C.,more preferably 42-44° C., until the desired weight gain (coatingweight) has been achieved.

Subjects, Patients and Patient Groups

The subject/patient may be an animal, a mammal, a placental mammal, arodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., amouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine(e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine(e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate,simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), amonotreme (e.g. platypus), an ape (e.g., gorilla, chimpanzee, orangutan,gibbon), or a human.

In preferred embodiments, the subject/patient is a human who has beendiagnosed as having one of the cognitive or CNS disorders describedherein, or (for prophylactic treatment) assessed as being susceptible toone of the neurodegenerative disorders of protein aggregation (e.g.cognitive or CNS disorder) described herein—for example based onfamilial or genetic or other data.

The patient may be an adult human, and the dosages described herein arepremised on that basis (typical weight 50 to 70 kg). If desired,corresponding dosages may be utilised for subjects outside of this rangeby using a subject weight factor whereby the subject weight is dividedby 60 kg to provide the multiplicative factor for that individualsubject.

The low dosage treatments of the present invention increase thefeasibility of purely prophylactic treatments, since the reducedconcentration of active ingredients inevitably reduces risk of anyadverse side effects (and increases the safety profile) and henceincreases the risk/benefit ratio for such prophylactic treatments.

Thus, for example, for diagnosis of AD, and assessment of severity, theinitial selection of a patient may involve any one or more of: rigorousevaluation by experienced clinician; exclusion of non-AD diagnosis asfar as possible by supplementary laboratory and other investigations;objective evaluation of level of cognitive function usingneuropathologically validated battery.

Diagnosis of AD and other disorders described herein can be performed byphysicians by methods well known to those skilled in the art.

As explained herein, in the present invention it is preferred that thesubject or patient group, if they are being treated in respect of AD, isone who is not receiving treatment with any of: an acetylcholinesteraseinhibitor or an N-methyl-D-aspartate receptor antagonist. Examples ofacetylcholinesterase inhibitors include Donepezil (Aricept™),Rivastigmine (Exelon™) or Galantamine (Reminyl™). An examples of an NMDAreceptor antagonist is Memantine (Ebixa™, Namenda™).

Based on the findings described herein, these selection criteria arelikely to be applicable when treating AD with MT⁺ compounds even athigher doses than those described herein e.g. between 0.5 mg and 300 mgtotal daily dose per day.

Thus in one aspect the present invention provides a method of treatment(or prophylaxis) of AD in a subject,

-   -   which method comprises orally administering to said subject a        methylthioninium (MT) containing compound,    -   with the proviso that said treatment does not include        administration of either or both of an acetylcholinesterase        inhibitor or an N-methyl-D-aspartate receptor antagonist.

Unless context demands otherwise, the disclosure made herein regardingthe “low dose” treatments of neurodegenerative diseases more generallyapplies mutatis mutandis to these aspects of the invention.

For example the AD subject or patient group may be entirely naïve tothese other treatments, and have not historically received one or bothof them.

For example the AD subject or patient group may have historicallyreceived one or both of them, but ceased that medication at least 1, 2,3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks, or morepreferably at least 1, 2, 3, 4, 5 or 6 months etc. prior to treatmentwith an MT compound according to the present invention.

Any aspect of the present invention may include the active step ofselecting the AD subject or patient group according to these criteria,or selecting an AD subject or patient group who is or are receivingtreatment with either or both an acetylcholinesterase inhibitor or anN-methyl-D-aspartate receptor antagonist, and discontinuing thattreatment (instructing the subject or patient group to discontinue thattreatment) prior to treatment with an MT compound according to thepresent invention.

Labels, Instructions and Kits of Parts

The unit dosage compositions described herein (e.g. a low dose MTcontaining compound plus optionally other ingredients, or MT compositionmore generally for treatment in AD) may be provided in a labelled packetalong with instructions for their use.

In one embodiment, the pack is a bottle, such as are well known in thepharmaceutical art. A typical bottle may be made from pharmacopoeialgrade HDPE (High-Density Polyethylene) with a childproof, HDPE pushlockclosure and contain silica gel desiccant, which is present in sachets orcanisters. The bottle itself may comprise a label, and be packaged in acardboard container with instructions for us and optionally a furthercopy of the label.

In one embodiment, the pack or packet is a blister pack (preferably onehaving aluminium cavity and aluminium foil) which is thus substantiallymoisture-impervious. In this case the pack may be packaged in acardboard container with instructions for us and label on the container.

Said label or instructions may provide information regarding theneurodegenerative disorders of protein aggregation (e.g. cognitive orCNS disorder) for which the medication is intended.

Where the medication is indicated for AD, said label or instructions mayprovide information instructing the user that the compositions shouldnot be used in conjunction with any of: an acetylcholinesteraseinhibitor or an N-methyl-D-aspartate receptor antagonist.

Said label or instructions may provide information regarding the maximumpermitted daily dosage of the compositions as described herein—forexample based on once daily, b.i.d., or t.i.d.

Said label or instructions may provide information regarding thesuggested duration of treatment, as described herein.

Reversing and/or Inhibiting the Aggregation of a Protein

One aspect of the invention is the use of an MT compound or compositionas described herein, to regulate (e.g., to reverse and/or inhibit) theaggregation of a protein, for example, aggregation of a proteinassociated with a neurodegenerative disease and/or clinical dementia.The aggregation will be associated with a disease state as discussedbelow.

Similarly, one aspect of the invention pertains to a method ofregulating (e.g., reversing and/or inhibiting) the aggregation of aprotein in the brain of a mammal, which aggregation is associated with adisease state as described herein, the treatment comprising the step ofadministering to said mammal in need of said treatment, aprophylactically or therapeutically effective amount of an MT compoundor composition as described herein, that is an inhibitor of saidaggregation.

Disease conditions treatable via the present invention are discussed inmore detail below.

Methods of Treatment

Another aspect of the present invention, as explained above, pertains toa method of treatment comprising administering to a patient in need oftreatment a prophylactically or therapeutically effective amount of acompound as described herein, preferably in the form of a pharmaceuticalcomposition.

Use in Methods of Therapy

Another aspect of the present invention pertains to a compound orcomposition as described herein, for use in a method of treatment (e.g.,of a disease condition) of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of an MTcompound or composition as described herein, in the manufacture of amedicament for use in treatment (e.g., of a disease condition).

In some embodiments, the medicament is a composition e.g. a low-doseunit dose composition as described herein.

Diseases of Protein Aggregation

The compounds and compositions of the present invention are useful inthe treatment or prophylaxis of diseases of protein aggregation.

Thus, in some embodiments, the disease condition is a disease of proteinaggregation, and, for example, the treatment is with an amount of acompound or composition as described herein, sufficient to inhibit theaggregation of the protein associated with said disease condition.

The following Table lists various disease-associated aggregatingproteins and the corresponding neurodegenerative disease of proteinaggregation. The use of the compounds and compositions of the inventionin respect of these proteins or diseases is encompassed by the presentinvention.

Diseases of protein aggregation Aggregating domain and/or Fibril subunitProtein Disease mutations size (kDa) Reference Neurodegenerativedisorders Prion protein Prion diseases Inherited and 27 Prusiner (1998)sporadic forms (CJD, nvCJD, Fatal PrP-27-30; many 27 Prusiner (1998)familial insomnia, mutations. Gerstmann-Straussler- Scheinker syndrome,Kuril) Fibrillogenic Gasset et al. (1992) domains: 113- 120, 178-191,202-218. Tau protein Alzheimer's disease, Inherited and 10-12 Wischik etal. (1988) Down's syndrome, FTDP- sporadic forms 17, CBD,post-encephalitic parkinsonism, Pick's disease, parkinsonism withdementia complex of Guam Truncated tau 10-12 Wischik et al. (1988)(tubulin-binding domain) 297-391. Mutations in tau Hutton et al. (1998)in FTDP-17. Many mutations Czech et al. (2000) in presenilin proteins.Amyloid Alzheimer's disease, Inherited and  4 Glenner & Wong, β-proteinDown's syndrome sporadic forms (1984) Amyloid β-  4 Glenner & Wong,protein; 1-42(3). (1984) Mutations in APP Goate et al. (1991) in rarefamilies. Huntingtin Huntington's disease N-termini of 40 DiFiglia etal. (1997) protein with expanded glutamine repeats. Ataxin)Spinocerebellar ataxias Proteins with Paulson et al. (1999) (SCA1, 2, 3,7) expanded glutamine repeats. Atrophin DentatorubropallidoluysianProteins with Paulson et al. (1999) atrophy (DRPLA) expanded glutaminerepeats. Androgen Spinal and bulbar Proteins with Paulson et al. (1999)receptor muscular atrophy expanded glutamine repeats. NeuroserpinFamilial encephalopathy Neuroserpin; 57 Davis et al. (1999) withneuronal inclusion S49P, S52R. bodies (FENIB) α-Synuclein Parkinson'sdisease, Inherited and 19 Spillantini et al. dementia with Lewy sporadicforms (1998) also bodies, multiple system PCT/GB2007/001105 atrophyA53T, A30P in Polymeropoulos et rare autosomal- al. (1997) dominant PDfamilies. TDP-43 FTLD-TDP Several TDP-43 10-43 Mackenzie et al.mutations (2010) Amyotrophic lateral Several TDP-43 10-43 Mackenzie etal. sclerosis mutations (2010) Cystatin C Hereditary cerebral Cystatin Cless 12-13 Abrahamson et al. angiopathy (Icelandic) 10 residues; (1992)L68Q. Superoxide Amyotrophic lateral SOD1 mutations. 16 Shibata et al.(1996) dismutase 1 sclerosis

As described in WO 02/055720, WO2007/110630, and WO2007/110627,diaminophenothiazines have utility in the inhibition of such proteinaggregating diseases.

Thus it will be appreciated that, except where context requiresotherwise, description of embodiments with respect to tau protein ortau-like proteins (e.g., MAP2; see below), should be taken as applyingequally to the other proteins discussed herein (e.g., β-amyloid,synuclein, prion, etc.) or other proteins which may initiate or undergoa similar pathological aggregation by virtue of conformational change ina domain critical for propagation of the aggregation, or which impartsproteolytic stability to the aggregate thus formed (see, e.g., thearticle by Wischik et al. in “Neurobiology of Alzheimer's Disease”, 2ndEdition, 2000, Eds. Dawbarn, D. and Allen, S. J., The Molecular andCellular Neurobiology Series, Bios Scientific Publishers, Oxford). Allsuch proteins may be referred to herein as “aggregating diseaseproteins.”

Likewise, where mention is made herein of “tau-tau aggregation”, or thelike, this may also be taken to be applicable to other“aggregating-protein aggregation”, such as β-amyloid aggregation, prionaggregation, synuclein aggregation, etc. The same applies for “tauproteolytic degradation” etc.

Preferred Aggregating Disease Target Proteins

Preferred embodiments of the invention are based on tau protein. Theterm “tau protein,” as used herein, refers generally to any protein ofthe tau protein family. Tau proteins are characterised as being oneamong a larger number of protein families which co-purify withmicrotubules during repeated cycles of assembly and disassembly (see,e.g., Shelanski et al., 1973, Proc. Natl. Acad. Sci. USA, Vol. 70, pp.765-768), and are known as microtubule-associated-proteins (MAPs).Members of the tau family share the common features of having acharacteristic N-terminal segment, sequences of approximately 50 aminoacids inserted in the N-terminal segment, which are developmentallyregulated in the brain, a characteristic tandem repeat region consistingof 3 or 4 tandem repeats of 31-32 amino acids, and a C-terminal tail.

MAP2 is the predominant microtubule-associated protein in thesomatodendritic compartment (see, e.g., Matus, A., in “Microtubules”[Hyams and Lloyd, Eds.] pp. 155-166, John Wiley and Sons, New York,USA). MAP2 isoforms are almost identical to tau protein in the tandemrepeat region, but differ substantially both in the sequence and extentof the N-terminal domain (see, e.g., Kindler and Garner, 1994, Mol.Brain Res., Vol. 26, pp. 218-224). Nevertheless, aggregation in thetandem-repeat region is not selective for the tau repeat domain. Thus itwill be appreciated that any discussion herein in relation to tauprotein or tau-tau aggregation should be taken as relating also totau-MAP2 aggregation, MAP2-MAP2 aggregation, and so on.

In some embodiments, the protein is tau protein.

In some embodiments, the protein is a synuclein, e.g., α- orβ-synuclein.

In some embodiments, the protein is TDP-43.

TAR DNA-Binding Protein 43 (TDP-43) is a 414 amino acid protein encodedby TARDBP on chromosome 1p36.2. The protein is highly conserved, widelyexpressed, and predominantly localised to the nucleus but can shuttlebetween the nucleus and cytoplasm (Mackenzie et al 2010). It is involvedin transcription and splicing regulation and may have roles in otherprocesses, such as: microRNA processing, apoptosis, cell division,stabilisation of messenger RNA, regulation of neuronal plasticity andmaintenance of dendritic integrity. Furthermore, since 2006 asubstantial body of evidence has accumulated in support of the TDP-43toxic gain of function hypothesis in amyotrophic lateral sclerosis(ALS). TDP-43 is an inherently aggregation-prone protein and aggregatesformed in vitro are ultrastructurally similar to the TDP-43 depositsseen in degenerating neurones in ALS patients (Johnson et al 2009).Johnson et al (2008) showed that when TDP-43 is overexpressed in a yeastmodel only the aggregated form is toxic. Several in vitro studies havealso shown that C-terminal fragments of TDP-43 are more likely thanfull-length TDP-43 to form insoluble cytoplasmic aggregates that becomeubiquitinated, and toxic to cells (Arai et al 2010; Igaz et al 2009;Nonaka et al 2009; Zhang et al 2009). Though Nonaka et al (2009)suggested that these cytoplasmic aggregates bind the endogenousfull-length protein depleting it from the nucleus, Zhang et al (2009)found retention of normal nuclear expression, suggesting a purely toxiceffect for the aggregates. Yang et al (2010) have described the captureof full-length TDP-43 within aggregates of C- and N-terminal fragmentsof TDP-43 in NSC34 motor neurons in culture. Neurite outgrowth, impairedas a result of the presence of such truncated fragments, could berescued by overexpression of the full-length protein. Although the roleof neurite outgrowth in vivo has not been established, this model wouldsupport the suggestion made by Nonaka and colleagues for a role ofTDP-43 aggregation in ALS pathogenesis.

Mutant TDP-43 expression in cell cultures has repeatedly been reportedto result in increased generation of C-terminal fragments, with evengreater cytoplasmic aggregation and toxic effects than the wild-typeprotein (Kabashi et al 2008; Sreedharan et al 2008; Johnson et al 2009;Nonaka et al 2009; Arai et al 2010; Barmarda et al 2010; Kabashi et al2010).

Where the protein is tau protein, in some embodiments of the presentinvention, there is provided a method of inhibiting production ofprotein aggregates (e.g. in the form of paired helical filaments (PHFs),optionally in neurofibrillary tangles (NFTs) in the brain of a mammal,the treatment being as described above.

Preferred Indications—Diseases of Protein Aggregation

In one embodiment the present invention is used for the treatment ofAlzheimer's disease (AD)—for example mild, moderate or severe AD.

Notably it is not only Alzheimer's disease (AD) in which tau protein(and aberrant function or processing thereof) may play a role. Thepathogenesis of neurodegenerative disorders such as Pick's disease andprogressive supranuclear palsy (PSP) appears to correlate with anaccumulation of pathological truncated tau aggregates in the dentategyrus and stellate pyramidal cells of the neocortex, respectively. Otherdementias include frontotemporal dementia (FTD); FTD with parkinsonismlinked to chromosome 17 (FTDP-17);disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration(CBD) and others (see, e.g., the article by Wischik et al. in“Neurobiology of Alzheimer's Disease”, 2nd Edition, 2000, Eds. Dawbarn,D. and Allen, S. J., The Molecular and Cellular Neurobiology Series,Bios Scientific Publishers, Oxford; especially Table 5.1). All of thesediseases, which are characterized primarily or partially by abnormal tauaggregation, are referred to herein as “tauopathies”.

Thus, in some embodiments, the disease condition is a tauopathy.

In some embodiments, the disease condition is a neurodegenerativetauopathy.

In some embodiments, the disease condition is selected from Alzheimer'sdisease (AD), Pick's disease, progressive supranuclear palsy (PSP),fronto temporal dementia (FTD), FTD with parkinsonism linked tochromosome 17 (FTDP 17), frontotemporal lobar degeneration (FTLD)syndromes; disinhibition-dementia-parkinsonism-amyotrophy complex(DDPAC), pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome,pallido nigro luysian degeneration (PNLD), cortico-basal degeneration(CBD), dementia with argyrophilic grains (AgD), dementia pugilistica(DP) or chronic traumatic encephalopathy (CTE), Down's syndrome (DS),dementia with Lewy bodies (DLB), subacute sclerosing panencephalitis(SSPE), MCI, Niemann-Pick disease, type C (NPC), Sanfilippo syndrometype B (mucopolysaccharidosis III B), or myotonic dystrophies (DM), DM1or DM2, or chronic traumatic encephalopathy (CTE).

In some embodiments, the disease condition is a lysosomal storagedisorder with tau pathology. NPC is caused by mutations in the geneNPC1, which affects cholesterol metabolism (Love et al 1995) andSanfilippo syndrome type B is caused by a mutation in the gene NAGLU, inwhich there is lysosomal accumulation of heparin sulphate (Ohmi et al.2009). In these lysosomal storage disorders, tau pathology is observedand its treatment may decrease the progression of the disease. Otherlysosomal storage disorders may also be characterised by accumulation oftau.

Use of phenothiazine diaminium salts in the treatment of Parkinson'sdisease and MCI is described in more detail in PCT/GB2007/001105 andPCT/GB2008/002066.

In some embodiments, the disease condition is Parkinson's disease, MCI,or Alzheimer's disease.

In some embodiments, the disease condition is Huntington's disease orother polyglutamine disorder such as spinal bulbar muscular atrophy (orKennedy disease), and dentatorubropallidoluysian atrophy and variousspinocerebellar ataxias.

In some embodiments, the disease condition is an FTLD syndrome (whichmay for example be a tauopathy or TDP-43 proteinopathy, see below).

In some embodiments, the disease condition is PSP or ALS.

TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS;ALS-TDP) and frontotemporal lobar degeneration (FTLD-TDP).

The role of TDP-43 in neurodegeneration in ALS and otherneurodegenerative disorders has been reviewed in several recentpublications (Chen-Plotkin et al 2010; Gendron et al 2010; Geser et al2010; Mackenzie et al 2010).

ALS is a neurodegenerative disease, characterised by progressiveparalysis and muscle wasting, consequent on the degeneration of bothupper and lower motor neurones in the primary motor cortex, brainstemand spinal cord. It is sometimes referred to as motor neuron disease(MND) but there are diseases other than ALS which affect either upper orlower motor neurons. A definite diagnosis requires both upper and lowermotor neurone signs in the bulbar, arm and leg musculature with clearevidence of clinical progression that cannot be explained by any otherdisease process (Wijesekera and Leigh 2009).

Although the majority of cases are ALS-TDP, there are other cases wherethe pathological protein differs from TDP-43. Misfolded SOD1 is thepathological protein in ubiquitin-positive inclusions in ALS with SOD1mutations (Seetharaman et al 2009) and in a very small subset(approximately 3-4%) of familial ALS, due to mutations in FUS (fused insarcoma protein), the ubiquitinated pathological protein is FUS (Vanceet al 2009; Blair et al 2010). FUS, like TDP-43, appears to be importantin nuclear-cytoplasmic shuttling although the ways in which impairednuclear import of FUS remains unclear. A new molecular classification ofALS, adapted from Mackenzie et al (2010), reflects the distinctunderlying pathological mechanisms in the different subtypes (see Tablebelow).

New Molecular Classification of ALS (modified from Mackenzie et al2010). In the majority of cases, TDP-43 is the pathologicalubiquitinated protein found in ALS.

Ubiquitin-positive inclusions in ALS Ubiquitinated disease proteinTDP-43 FUS SOD1 Clinico- ALS-TDP ALS-FUS ALS-SOD1 pathologic subtypeAssociated TARDBP FUS SOD1 genotype Frequency of Common Rare Rare ALScases

Amyotrophic lateral sclerosis has been recognised as a nosologicalentity for almost a century and a half and it is recognised in ICD-10 isclassified as a subtype of MND in ICD 10 (G12.2). Reliable clinicaldiagnostic are available for ALS, which differ little from Charcot'soriginal description, and neuropathological criteria, reflecting theunderlying molecular pathology, have also been agreed.

While ALS is classified pathologically into three subgroups, ALS-TDP,ALS-SOD1 and ALS-FUS, both latter conditions are rare. The largest studyto date showed all sporadic ALS cases to have TDP-43 pathology(Mackenzie et al 2007). Only around 5% of ALS is familial (Byrne et al2010) and mutations in SOD1, the commonest mutations found in FALS,account for between 12-23% of cases (Andersen et al 2006). SOD1 may alsobe implicated in 2-7% of SALS. Mutations in FUS appear to be far lesscommon, accounting for only around 3-4% of FALS (Blair et al 2010). Soit can be reliably predicted that a clinical case of SALS will haveTDP-43 based pathology. Similarly this can be reliably predicted in FALSdue to mutations in TDP-43, which account for around 4% of cases(Mackenzie et al 2010). ALS with mutations in: VCP, accounting for 1-2%of FALS (Johnson et al 2010), ANG (Seilhean et al 2009), and CHMP2B (Coxet al 2010) have also been reported to be associated with TDP-43positive pathology. Although SOD1, FUS and ATXN2 mutations have not beenfound to be associated with TDP-43 positive aggregates, it has howeverbeen reported that TDP-43 is implicated in the pathological processesputatively arising from these mutations (Higashi et al 2010; Ling et al2010; Elden et al 2010).

It is therefore established that TDP-43 has an important, andpotentially central role, in the pathogenesis of the vast majority ofSALS cases and may be implicated in the pathogenesis of a significantproportion of FALS. ALS is now widely considered to be a TDP-43proteinopathy (Neumann et al 2009) and numerous in vitro, and in vivostudies provide support to the hypothesis that toxic gain of function,due to TDP-43 aggregation is responsible for at least some of theneurotoxicity in the disease.

FTLD syndromes are insidious onset, inexorably progressive,neurodegenerative conditions, with peak onset in late middle age. Thereis often a positive family history of similar disorders in a firstdegree relative.

Behavioural variant FTD is characterised by early prominent change insocial and interpersonal function, often accompanied by repetitivebehaviours and changes in eating pattern. In semantic dementia there areprominent word finding problems, despite otherwise fluent speech, withdegraded object knowledge and impaired single word comprehension oncognitive assessment. Progressive non-fluent aphasia presents with acombination of motor speech problems and grammatical deficits. The coreclinical diagnostic features for these three FTLD syndromes are shown inthe Table below and the full criteria in Neary et al (1998).

Clinical Profile and Core Diagnostic Features of FTLD Syndromes

FTLD Syndrome -Clinical Profile Core Diagnostic Features FrontotemporalDementia 1. Insidious onset and gradual Character change and disorderedsocial progression conduct are the dominant features initially 2. Earlydecline in social interpersonal and throughout the disease course.conduct Instrumental functions of perception, 3. Early impairment inregulation of spatial skills, praxis and memory are intact personalconduct or relatively well preserved. 4. Early emotional blunting 5.Early loss of insight Semantic Dementia A) Insidious onset and gradualprogression Semantic disorder (impaired B) Language disordercharacterised by understanding of word meaning and/or 1. Progressive,fluent empty speech object identity) is the dominant feature 2. Loss ofword meaning manifest by initially and throughout the disease course.impaired naming and Other aspects of cognition, including comprehensionautobiographic memory, are intact or 3. Semantic paraphasias and/orrelatively well preserved. 4. Perceptual disorder characterised by 1.Prosopagnosia: impaired recognition of identity of familiar faces and/or2. Associative agnosia: impaired recognition of object identity C)Preserved perceptual matching and drawing reproduction D) Preservedsingle word repetition E) Preserved ability to read aloud and write todictation orthographically regular words Progressive Non-fluent AphasiaA) Insidious onset and gradual Disorder of expressive language is theprogression dominant feature initially and throughout B) Non-fluentspontaneous speech with at the disease course. Other aspects of leastone of the following: cognition are intact or relatively wellagrammatism, phonemic paraphasias preserved. or anomia

The discovery that TDP-43-positive inclusions characterize ALS andFTLD-TDP (Neumann et al 2006) was quickly followed by the identificationof missense mutations in the TARDBP gene in both familial and sporadiccases of ALS (Gitcho et al 2008; Sreedharan et al., 2008). So far, 38different TARDBP mutations have been reported in 79 genealogicallyunrelated families worldwide (Mackenzie et al 2010). TARDBP mutationsaccount for approximately 4% of all familial and around 1.5% of sporadicALS cases.

As of December 2010, mutations in thirteen genes which are associatedwith familial and sporadic ALS have been identified. Linkage of ALS tofive other chromosome loci has been demonstrated but thus far specificmutations have not been identified.

TDP-43 Proteinopathies

MT has a mode of action which targets and can reduce TDP-43 proteinaggregation in cells, which is a pathological feature of the vastmajority of both familial and sporadic ALS and is also characteristic ofFTLD-P.

In addition laboratory data shows that methylthioninium inhibits theformation of TDP-43 aggregates in SH-SY5Y cells. Following treatmentwith 0.05 μM MT, the number of TDP-43 aggregates was reduced by 50%.These findings were confirmed by immunoblot analysis (Yamashita et al2009).

The compounds and compositions of the invention may therefore be usefulfor the treatment of amyotrophic lateral sclerosis (ALS) andfrontotemporal lobar degeneration (FTLD).

Huntington's Disease and Polyglutamine Disorders

MT can reduce polyglutamine protein aggregation in cells, which is apathological feature of Huntington's disease. Huntington's disease iscaused by expansion of a translated CAG repeat located in the N-terminusof huntingtin. Wild-type chromosomes contain 6-34 repeats whereas, inHuntington's disease, chromosomes contain 36-121 repeats. The age ofonset of disease correlates inversely with the length of the CAG tractsthat code for polyglutamine repeats within the protein.

Laboratory data shows that methylthioninium inhibits the formation ofaggregates of a huntingtin derivative containing a polyglutamine stretchof 102 residues in zebrafish (van Bebber et al. 2010). MT, when testedat 0, 10 and 100 μM, prevented the formation of such aggregates inzebrafish in a dose dependent manner.

The compounds and compositions of the invention may therefore be usefulfor the treatment of Huntington's disease and other polyglutaminedisorders such as spinal bulbar muscular atrophy (or Kennedy disease),and dentatorubropallidoluysian atrophy and various spinocerebellarataxias (Orr & Zoghbi, 2007).

Mitochondrial Diseases and Lafora Disease

The organ most frequently affected in mitochondrial disorders,particularly respiratory chain diseases (RCDs), in addition to theskeletal muscle, is the central nervous system (CNS). CNS manifestationsof RCDs comprise stroke-like episodes, epilepsy, migraine, ataxia,spasticity, movement disorders, psychiatric disorders, cognitivedecline, or even dementia (mitochondrial dementia). So far mitochondrialdementia has been reported in MELAS, MERRF, LHON, CPEO, KSS, MNGIE,NARP, Leigh syndrome, and Alpers-Huttenlocher disease (Finsterer, 2009).There are four complexes in the mitochondrial respiration chain,involving a series of electron transfers. Abnormal function of any ofthese complexes can result in mitochondrial diseases secondary to anabnormal electron transport chain and subsequent abnormal mitochondrialrespiration. Complex III of the mitochondrial respiration chain acts totransfer electrons to cytochrome c.

Compounds and compositions of the invention may also be used to treatmitochondrial diseases which are associated with a deficient and/orimpaired complex III function of the respiration chain. The compoundshave the ability to act as effective electron carrier and/or transfer,as the thioninium moiety has a low redox potential converting betweenthe oxidised and reduced form. In the event of an impaired and/ordeficient function of Complex III leading to mitochondrial diseases,compounds of the invention are also able to perform the electrontransportation and transfer role of complex III because of the abilityof the thioninium moiety to shuttle between the oxidised and reducedform, thus acting as an electron carrier in place of sub-optimallyfunctioning complex III, transferring electrons to cytochrome c.

Compounds and compositions of the invention also have the ability togenerate an active thioninium moiety that has the ability to divertmisfolded protein/amino acid monomers/oligomers away from the Hsp70ADP-associated protein accumulation and/or refolding pathways, andinstead rechannel these abnormal folded protein monomers/oligomers tothe pathway that leads directly to the Hsp70 ATP-dependentubiquitin-proteasome system (UPS), a pathway which removes thesemisfolded proteins/amino acid monomers/oligomers via the direct route(Jinwal et al. 2009).

Lafora disease (LD) is an autosomal recessive teenage-onset fatalepilepsy associated with a gradual accumulation of poorly branched andinsoluble glycogen, termed polyglucosan, in many tissues. In the brain,polyglucosan bodies, or Lafora bodies, form in neurons. Inhibition ofHsp70 ATPase by MT (Jinwal et al. 2009) may upregulate the removal ofmisfolded proteins. Lafora disease is primarily due to a lysosomalubiquitin-proteasomal system (UPS) defect because of a mutation ineither the Laforin or Malin genes, both located on Chromosome 6, whichresult in inclusions that may accelerate the aggregation of misfoldedtau protein. Secondary mitochondrial damage from the impaired UPS mayfurther result in a suppressed mitochondrial activity and impairedelectron transport chain leading to further lipofuscin and initiatingthe seizures that are characteristic of Lafora disease.

The MT moiety may disaggregate existing tau aggregates, reduce more tauaccumulating and enhance lysosomal efficiency by inhibiting Hsp70ATPase. MT may lead to a reduction in tau tangles by enhancing theubiquitin proteasomal system removal of tau monomers/oligomers, throughits inhibitory action on Hsp70 ATPase.

Thus compounds and compositions of the present invention may haveutility in the treatment of Lafora disease.

Mixtures of Oxidised and Reduced MT Compounds

MT compounds for use in the present invention may include mixtures ofthe oxidised and reduced form.

In particular, the LMT-containing compounds may include oxidised (MT⁺)compounds as ‘impurities’ during synthesis, and may also oxidize (e.g.,autoxidize) after synthesis to give the corresponding oxidized forms.Thus, it is likely, if not inevitable, that compositions comprising thecompounds of the present invention will contain, as an impurity, atleast some of the corresponding oxidized compound. For example an “LMT”salt may include 10 to 15% of MT⁺ salt.

When using mixed MT compounds the MT dose can be readily calculatedusing the molecular weight factors of the compounds present.

Salts and Solvates

Although the MT containing compounds described herein are themselvessalts, they may also be provided in the form of a mixed salt (i.e., thecompound of the invention in combination with another salt). Such mixedsalts are intended to be encompassed by the term “and pharmaceuticallyacceptable salts thereof”. Unless otherwise specified, a reference to aparticular compound also includes salts thereof.

The compounds of the invention may also be provided in the form of asolvate or hydrate. The term “solvate” is used herein in theconventional sense to refer to a complex of solute (e.g., compound, saltof compound) and solvent. If the solvent is water, the solvate may beconveniently referred to as a hydrate, for example, a mono-hydrate, adi-hydrate, a tri-hydrate, a penta-hydrate etc. Unless otherwisespecified, any reference to a compound also includes solvate and anyhydrate forms thereof.

Naturally, solvates or hydrates of salts of the compounds are alsoencompassed by the present invention.

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Any sub-titles herein are included for convenience only, and are not tobe construed as limiting the disclosure in any way.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may beused by those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

FIGURES

FIG. 1 . (A) Screening, safety and efficacy populations. (B) Patientcategorisation by randomised treatment, AD-approved co-medication statusand withdrawal rates at 65 weeks.

FIG. 2 . Least squares estimates of change from baseline in (A) ADAS-cogand (B) ADCS-ADL using prespecified repeat of primary analysis withstratification factor AChEI/Mem as an interaction term in the model.

FIG. 3 . Least squares estimates of change from baseline for (A)ADAS-cog and (B) ADCS-ADL in patients restricted to those randomised tothe control arm and receiving 4 mg b.i.d. of LMTM, either alone or incombination with AD-labelled medications.

FIG. 4 . Least squares estimates of LVV change over time usingprespecified repeat of primary analysis with stratification co-variateAChEI/Mem as an interaction term in the model.

FIG. 5 . Least squares estimates of TPV change from baseline usingprespecified repeat of primary analysis with stratification factorAChEI/Mem as an interaction term in the model.

FIG. 6 . Least squares estimates of HV change from baseline usingprespecified repeat of primary analysis with stratification factorAChEI/Mem as an interaction term in the model.

FIG. 7 . Least squares estimates of responses for change from baselinein (A) LVV, (B) TPV and (C) HV in subjects restricted to thoserandomised to the control arm and receiving 4 mg b.i.d. of LMTM eitheralone or in combination with AD-labelled medications.

EXAMPLES Example 1—Provision of MT-Containing Compounds

Methods for the chemical synthesis of the MT-containing compoundsdescribed herein are known in the art. For example:

Synthesis of compounds 1 to 7 can be performed according to the methodsdescribed in WO2012/107706, or methods analogous to those.

Synthesis of compound 8 can be performed according to the methodsdescribed in WO2007/110627, or a method analogous to those.

Synthesis of compound 9 (MTC) is well known in the art. Examplessyntheses of highly pure MTC are provided in WO2006/032879 andWO2008/007074.

Synthesis of compounds 10 to 13 can be performed according to themethods described in WO2007/110630, or methods analogous to those.

Example 2—Formulation of MT-Containing Compounds

Methods for the chemical synthesis of the MT-containing compoundsdescribed herein are known in the art. Example methods using drycompression, for example, are provided in WO2012/072977.

Example 3—Phase 3 Clinical Trial in Mild to Moderate AD

Methods

Outcomes and Measures

The co-primary efficacy outcomes were change from baseline in 11-itemAlzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) andthe 23-item Alzheimer's Disease Cooperative Study Activities of DailyLiving (ADCS-ADL). Magnetic resonance imaging (MRI) volumetry was chosenas the principal secondary outcome to support a potential effect on therate of brain atrophy (Fox et al., 1997; Ridha et al., 2008). Change inLateral Ventricular Volume (LVV), as measured by the VentricularBoundary Shift Integral (VBSI), (Salloway et al., 2014) was chosen asthe principal measure as it provides high-contrast boundaries formeasurement and is less affected by motion artefacts than whole brain orsmaller structures. Key supportive topographical measures(temporo-parietal (TPV) and hippocampal volume (HV)) are also reported.

Patients

Patients were recruited at 115 sites across 16 countries between January2013 and August 2014. Enrolment of approximately 833 patients wastargeted, with 891 patients actually recruited, in order to obtain dataon approximately 500 patients completing the study, assuming a 30-40%drop-out rate. Patients aged <90 meeting a diagnosis of all causedementia and probable AD according to National Institute of Aging (NIA)and Alzheimer's Association (AA) criteria were included if they had aClinical Dementia Rating (CDR) total score of 1 or 2 and Mini-MentalState Examination (MMSE) score of 14-26 inclusive. Adult caregiver(s)were required to participate. Concomitant use of AChEIs and/or memantinewas permitted, provided the patient had been taking the medication(s)for ≥3 months, with no changes to the dosage for weeks prior toscreening. Concomitant use of serotonergic antidepressant medication waspermitted, but patients were monitored closely using a targeted ratingscale derived from 4 published diagnostic criteria (Alusik et al., 2014)due to a theoretical potential for serotonin syndrome (Ramsay et al.,2007). Patients were excluded from the study if they had a significantcentral nervous system disorder other than AD or significant focal orvascular intracranial pathology on brain magnetic resonance imaging(MRI) performed within 6 weeks prior to baseline. Because MT⁺ in highdoses can induce methaemoglobinaemia, subjects with glucose-6-phosphatedehydrogenase deficiency or who were otherwise at haematological riskwere excluded. Other inclusion/exclusion criteria are provided in theSupplementary Materials.

Randomisation and Masking

Patients were randomised at baseline to LMTM 75 mg b.i.d. or 125 mgb.i.d. (expressed as MT base equivalent) or control in a 3:3:4 ratio.The randomisation was stratified according to geographical region (3levels: North America, Europe, rest of world), use of AD-labelledco-medications (2 levels, using or not using) and severity (2 levels,moderate MMSE 14-19 inclusive, mild MMSE 20-26 inclusive). Patients inthe control arm received a dose of 4 mg b.i.d. to maintain blinding.

Ethical Conduct of the Study

All patients provided written informed consent prior to enrolling in thestudy; legally acceptable representatives provided consent on behalf ofpatients with reduced decision-making capacity. Adult caregivers alsoprovided consent for their own involvement. The study was conducted inaccordance with the Declaration of Helsinki and the InternationalConference on Harmonisation Guidelines for Good Clinical Practice, andapproval of the study protocol and all related documents was obtainedfrom the appropriate Independent Ethics Committees and InstitutionalReview Boards for all study sites. An independent Data and SafetyMonitoring Board was established for oversight of accruing safetyinformation. The trial is registered at www.clinicaltrials.gov(NCT01689246) and the European Union Clinical Trials Registry(2012-002866-11).

Clinical and Imaging Assessments

ADAS-cog and ADCS-ADL assessments were performed at baseline and every13 weeks thereafter, with change at 65 weeks (the final on-treatmentvisit) the primary efficacy measures. These were repeated at the finaloff-treatment follow-up visit at Week 69. Secondary efficacy measuresincluded Clinical Global Impression of Change (ADCS-CGIC, administeredby an independent rater); MMSE, administered on screening and at Weeks26, 52, 65 and 69. Brain MRI scans were performed at baseline/screeningand every 13 weeks using a standardized protocol including volumetric 3DT1-weighted data consistent with ADNI recommendations, FLAIR, T2*Gradient Echo and T2-weighted sequences. Diffusion-Weighted Imaging wasalso available at screening in order to exclude patients withco-existing pathology that would lead to a diagnosis other than probableAD. Each site was first qualified ensuring standardised acquisitionprotocol, patient handling and data management. MRI data were centrallycollected by Imaging Corelab (Bioclinica). Data were centrally reviewedby RadMD for eligibility and safety (Amyloid Related ImagingAbnormalities, or ARIA monitoring) on an on-going basis. Volumetric 3DT1data were also reviewed centrally in order to measure (using a boundaryshift integral (BSI) technique; Salloway et al., 2014) change in LVV asprincipal secondary outcome measure, and as exploratory endpointstemporo-parietal volume (TPV), whole brain volume (WBV) and hippocampalvolume (HV, estimated as the mean of left and right) (Wischik et al.,2016). Additional exploratory endpoints included change in glucoseuptake in the temporal lobe, assessed using ¹⁸F-fluorodeoxyglucosepositron emission tomography (FDG-PET) performed during screening and atWeeks 39 and 65 in a subset of patients in sites with this imagingcapability. Changes in cerebrospinal fluid biomarkers of Alzheimer'sdisease, including total tau, phospho-tau and amyloid-β₁₋₄₂, wereexplored using cerebrospinal fluid samples that were collected atbaseline and Week 65 (or early termination visit). Lumbar punctures wereperformed only in subjects who were themselves able to provide consentspecifically for this procedure.

Patients were monitored throughout for adverse events (AEs) and clinicallaboratory testing, physical and neurological examinations and 12-leadelectrocardiograms were performed at all clinic visits (screening,baseline and Weeks 2, 6, 13, 26, 39, 52, 65 and 69). Patients were alsoassessed at all visits for suicidal ideation and intent, and weresystematically assessed for signs of serotonin toxicity.

Statistical Methods

The primary efficacy analyses of change from baseline in ADAS-cog andADCS-ADL scores were conducted in the modified intent-to-treat (mITT)population (all randomised patients who took at least one dose of studytreatment and had at least one post-baseline, on-treatment efficacyassessment). The primary analysis was specified as a mixed modelrepeated-measures (MMRM) analysis with an unstructured covariance matrixand no imputation for missing data. The model included categorical visit(5 levels corresponding to assessments at weeks 13, 26, 39, 52 and 65),treatment (3 levels corresponding to control, 75 mg b.i.d. and 125 mgb.i.d.), treatment-by-visit interaction, and the stratificationvariables as additive terms, and baseline ADAS-cog or ADCS-ADL as acovariate. A similar exploratory analysis was specified in theStatistical Analysis Plan (SAP) with the AChEI/Mem covariate as aninteraction term with treatment and as an interaction term with visit.Westfall's method for multiple comparison correction was used in eachstep to ensure strong control of the familywise error with alpha 0.05(Westfall et al., 1997) The same analyses were implemented for change inLVV, WBV, TPV, HV and TPV.

Results

Patients

The patient disposition and trial design is shown in FIG. 1 . Of 891patients randomised, 885 received at least one dose of study drug andcomprised the safety population, and 855 patients were in the mITTpopulation FIG. 1(A). Patient disposition by randomised treatment andtreatment with or without AChEI/Mem is shown in FIG. 1(B). The baselinedemographics and clinical characteristics of the safety population areshown in Table 1. There were 618 patients completing the study to 65weeks (with 579 remaining on treatment), an overall study withdrawalrate of 31%. MRI scans from all scheduled visits were available from 880patients pre-treatment and 554 at 65 weeks. FDG-PET data were availablefrom 101 patients at 65 weeks, of whom only 6 were not taking AChEI/Memtreatments.

TABLE 1 Patient baseline demographics and clinical characteristics(safety population) Control LMTM LMTM LMTM 4 mg b.i.d. 75 mg b.i.d. 125mg b.i.d. Total Characteristic n = 354 n = 267 n = 264 n = 885 Age(years) Mean (SD) 70.7 (8.5) 71.0 (9.3) 70.1 (9.3) 70.6 (9.0) Median(min; max) 72.0 (40; 89) 72.0 (39; 88) 71.0 (32; 89) 72.0 (32; 89) SexMale, n (%) 134 (38) 93 (35) 113 (43) 340 (38) Female, n (%) 220 (62)174 (65) 151 (57) 545 (62) Race American Indian or 2 (0.6) 3 (1.1) 2(0.8) 7 (0.8) Alaska Native, n (%) Asian, n (%) 41 (11.6) 32 (12.0) 30(11.4) 103 (11.6) Black or African 3 (0.8) 3 (1.1) 4 (1.5) 10 (1.1)American, n (%) White, n (%) 307 (86.7) 226 (84.6) 225 (85.2) 758 (85.6)Other, n (%) 1 (0.3)  0 2 (0.8) 3 (0.3) Multiple Race, n (%)  0 3 (1.1)1 (0.4) 4 (0.5) Years since diagnosis Mean (SD) 2.8 (2.4) 2.9 (2.3) 2.8(2.2) 2.8 (2.3) Dementia severity CDR 0.5, n (%) 4 (1.1) 1 (0.4) 2 (0.8)7 (0.8) CDR 1, n (%) 261 (73.7) 209 (78.3) 192 (72.7) 662 (74.8) CDR 2,n (%) 89 (25.1) 57 (21.3) 70 (26.5) 216 (24.4) MMSE Mean (SD) 18.6(3.45) 18.8 (3.44) 18.5 (3.40) 18.6 (3.43) Median (min; max) 18.0 (14;26) 19.0 (14; 26) 18.0 (14; 26) 18.0 (14; 26) MMSE severity MMSE ≥20, n(%) 134 (38) 105 (39) 98 (37) 337 (38) MMSE <20, n (%) 220 (62) 162 (61)166 (63) 548 (62) ADAS-Cog: Mean (SD) 27.2 (10.1) 26.5 (9.4) 26.7 (9.7)26.9 (9.8) Median (min; max) 26.3 (7; 57) 26.3 (8; 54) 26.3 (8; 56) 26.3(7; 57) ADCS-ADL: Mean (SD) 55.9 (12.7) 58.0 (11.1) 57.5 (12.7) 57.0(12.3) Median (min; max) 58.0 (17; 78) 58.5 (16; 78) 60.0 (13; 78) 59.0(13; 78) Whole brain volume (cm³) Mean (SD) 927 (108) 922 (115) 939(101) 929 (108) Median (min; max) 917 (681; 1,233) 922 (602; 1,207) 934(682; 1,264) 925 (602; 1,264) Lateral ventricular volume (cm³) Mean (SD)52 (23) 52 (26) 51 (23) 52 (24) Median (min; max) 49 (15; 154) 44 (12;160) 47 (15; 138) 47 (12; 160) Hippocampal volume (mm³) Mean (SD) 2.3(0.6) 2.7 (0.6) 2.9 (0.6) 2.8 (0.6) Median (min; max) 2.7 (1.4; 4.5) 2.7(1.4; 4.4) 2.8 (1.5; 5.0) 2.7 (1.4; 5.0) AD-approved co-medicationsAChEI only, n (%) 183 (52) 151 (57) 150 (57) 484 (55) Memantine only, n(%) 32 (9) 16 (6) 15 (6) 63 (7) AChEI and memantine, n (%) 93 (26) 60(23) 61 (23) 214 (24) CSF biomarkers (ng/L) Total tau, mean (SD) [n]143.9 (68.4) 156.4 (72.5) 113.2 (54.7) 144.8 (68.2) [19] [15] [5] [39]Phospho-tau, mean (SD) [n] 59.2 (25.3) 61.2 (20.3) 58.1 (12.8) 59.8(21.9) [20] [15] [5] [40] Aβ1-42, mean (SD) [n] 264.7 (96.6) 276.0(85.9) 235.8 (62.1) 265.3 (88.0) [20] [15] [5] [40] APOE genotype ε4allele present, n (%) 144 (47.5) 91 (41.9) 114 (52.5) 349 (47.4) ε4allele absent, n (%) 159 (52.5) 126 (58.1) 103 (47.5) 388 (52.6)

Efficacy Analyses of Primary Clinical Outcomes

Table 2 reports change in ADAS-cog and ADCS-ADL scores, decline in thecontrol arm and difference with respect to control by treatment arm. TheADAS-cog decline at 12 months (3.85±0.47, mean±se) was indistinguishablefrom a meta-analysis of recent studies (4.58, 95% CI: 3.69-5.47) at 12months (Salloway et al., 2014; Doody et al., 2014). Likewise, theADCS-ADL decline at 12 months (−6.51±0.63) was similar to that of theonly available recent study (Doody et al., 2014) in mild/moderate AD(−5.79, 95% CI: −7.10-−4.48).

As can be seen from Table 2, neither of the primary analyses yieldedsignificant effects for either 75 mg b.i.d. or for 125 mg b.i.d. at 65weeks. However, the AChEI/Mem factor was significant for both ADAS-cogand ADCS-ADL, with p-values 9.4e-05 and 0.0174 respectively aftercorrection for multiple comparisons. This implies a mean additivetreatment benefit in patients not taking AD treatments of −2.30 (95% CI:−3.35-−1.25) ADAS-cog units and 2.00 (95% CI: 0.65-3.35) ADCS-ADL units.Likewise, mild subjects had lower rates of progression for both ADAS-cogand ADCS-ADL overall (p values: 8.6e-04 and 9.6e-07, respectively).There was no overall effect of region on either outcome.

TABLE 2 Primary efficacy analyses for ADAS-cog (A) and ADCS-ADL (B) at65 weeks. In this and in all following tables, “pval-adj” signifies thep values corrected for multiple comparisons using the Westfallprocedure. All stratification covariates are additive terms. pval-Estimate SE 2.50% 97.50% p value adj ADAS-cog Control 6.32 0.52 5.317.34 75 mg b.i.d. −0.02 0.80 −1.60 1.56 0.983 0.983 125 mg b.i.d. −0.430.83 −2.06 1.20 0.602 0.932 Not using AChEI/ −2.30 0.53 −3.35 −1.251.78e−05 9.4e−05 Mem Mild −1.03 0.28 −1.57 −0.59 1.85e−04 8.6e−04 NorthAmerica −0.08 0.26 −0.58 0.43 0.769 0.947 Rest of world −0.47 0.44 −1.320.39 0.285 0.724 ADCS-ADL Control −8.22 0.72 −9.63 −6.82 75 mg b.i.d.−0.93 1.12 −3.12 1.26 0.407 0.866 125 mg b.i.d. −0.34 1.16 −2.61 1.930.770 0.948 Not using AChEI/ 2.00 0.69 0.65 3.35 0.00365 0.0174 Mem Mild1.62 0.31 1.02 2.23  2.0e−07 9.6e−07 North America 0.08 0.34 −0.58 0.740.814 0.948 Rest of world −0.27 0.56 −1.37 0.83 0.632 0.948

In order to understand better the role of the AChEI/Mem factor, apre-specified analysis was undertaken which included this as aninteraction term with visit and as an interaction term with LMTMtreatment in the model rather than only as an overall additive term asin the primary model. The results by dose-arm and AChEI/Mem status areshown in Table 3 and FIG. 2 for both ADAS-cog and ADCS-ADL.

TABLE 3 Prespecified repeat of primary analysis of ADAS-cog and ADCS-ADLwith stratification covariate AChEI/Mem as an interaction term in themodel. pval- Estimate SE 2.50% 97.50% p value adj ADAS-cog Control 5.980.51 5.00 6.98 0.000 75 mg b.i.d. −6.25 1.36 −8.92 −3.59 4.15e−061.5e−05 125 mg b.i.d. −5.79 1.37 −8.47 −3.11 2.35e−05 6.8e−05 75 mgb.i.d. + AChEI/ 1.02 0.81 −0.58 2.61 0.212 0.362 Mem 125 mg bi.d. +AChEI/ 0.50 0.84 −1.15 2.14 0.555 0.555 Mem ADCS-ADL Control −7.92 0.70−9.29 −6.55 0.000 75 mg b.i.d. 6.48 1.84 2.87 10.09  4.3e−04 0.00138 125mg b.i.d. 6.93 1.86 3.29 10.57  1.9e−04 0.00711 75 mg b.i.d. + AChEI/−2.16 1.13 −4.37 0.05 0.0553 0.103 Mem 125 mg bi.d. + AChEI/ −1.62 1.17−3.91 0.68 0.167 0.167 Mem

Given the effect of AD treatments on the efficacy of LMTM given athigher doses, the same effect was explored further in post-hoc analysesrestricted to subjects randomised to the control arm receiving 4 mgb.i.d. of LMTM. The results are shown in Table 4 and FIG. 3 for ADAS-cogand ADCS-ADL.

TABLE 4 Post-hoc analysis of ADAS-cog and ADCS-ADL restricted tosubjects randomised to the control arm and receiving 4 mg b.i.d. with orwithout AD-labelled medications. Estimate SE 2.50% 97.50% p valueADAS-cog 4 mg b.i.d. + 6.79 0.53 5.75 7.83 AChEI/Mem 4 mg b.i.d. −5.901.14 −8.13 −3.66 2.3e−07 ADCS-ADL 4 mg b.i.d. + −8.90 0.73 −10.34 −7.47AChEI/Mem 4 mg b.i.d. 7.19 1.55 4.15 10.23 3.5e−06

MRI Volumetric Analysis of Lateral Ventricular Volume (LVV),Temporo-Parietal Volume (TPV) and Hippocampal Volume (HV)

Using the same initial analysis model as for the primary clinicaloutcomes, no effect of treatment was seen (data not shown). In theprespecified analysis model with the AChEI/Mem factor as an interactionterm in the model, results similar to those shown for the clinicaloutcomes were found for LVV, with p values 4.0e-05 and 3.8e-04,respectively for the 75 mg b.i.d. or 125 mg b.i.d. doses. The resultswere similar in the TPV analysis, with p values 2.4e-04 and 0.00160 for75 mg b.i.d. and 125 mg b.i.d. doses respectively. In the HV analysis,only the 125 mg b.i.d. dose taken alone approached nominal significance(p=0.0558) in the mixed mild/moderate population. The data are shown inTable 5 and FIGS. 4, 5 and 6 .

TABLE 5 Prespecified repeat of primary analysis of LVV, TPV and HV (mm³)with stratification co-variate AChEI/Mem as an interaction term in themodel. pval- Estimate SE 2.50% 97.50% p value adj LVV Control 7,187 2796,641 7,733 75 mg b.i.d. −2,707 659 −3,999 −1,416 4.0e−05 1.9e−04 125 mgb.i.d. −2,347 660 −3,641 −1,052 3.8e−04 0.00124 75 mg b.i.d. + AChEI/−273 443 −1,141 596 0.538 0.733 Mem 125 mg b.i.d. + AChEI/ −306 453−1,194 583 0.500 0.733 Mem TPV Control −1529 46 −1620 −1438 75 mg b.i.d.472 117 244 701 5.2e−05 2.4e−04 125 mg b.i.d. 405 117 176 634 5.2e−040.00160 75 mg b.i.d. + AChEI/ −39 74 −183 106 0.597 0.825 Mem 125 mgb.i.d. + AChEI/ −12 75 −160 135 0.869 0.869 Mem HV Control −115.3 4.0−123.1 −108.5 75 mg b.i.d. 9.5 10.4 −10.8 29.8 0.360 0.571 125 mg b.i.d.20.0 10.4 −0.5 40.5 0.0558 0.187 75 mg b.i.d. + AChEI/ −2.0 6.4 −14.510.5 0.754 0.754 Mem 125 mg b.i.d. + AChEI/ −7.4 6.5 −20.2 5.3 0.2530.555 Mem

The effect of AD-labelled co-medications on the LVV, TPV and HV efficacyof low dose LMTM was explored further in the post-hoc analysisrestricted to subjects randomised to the control arm receiving LMTM 4 mgb.i.d. either alone or in combination with AChEI/Mem. The results areshown in Table 6 and FIG. 7 .

TABLE 6 Post-hoc analysis of LVV, TPV and HV (mm³) restricted tosubjects randomized to the control arm and receiving 4 mg b.i.d. of LMTMeither alone or in combination with AD-labelled medications. Estimate SE2.50% 97.50% p value LVV 4 mg b.i.d. + 7,426 289 6860 7,993 AChEI/Mem 4mg b.i.d. −1,894 549 −2,972 −818 0.000564 TPV 4 mg b.i.d. + −1,596 48−1,691 −1,501 AChEI/Mem 4 mg b.i.d. 534 98 342 725 4.9e−08 HV 4 mgb.i.d. + −117.8 4.1 −125.9 −109.7 AChEI/Mem 4 mg b.i.d. 19.5 8.7 2.336.6 0.0259 

Mild and Moderate AD

The same analyses were repeated in mild and moderate subjects separatelyas disease severity was a highly significant covariate in the primarymodel (Table 7). The results for treatment with LMTM as monotherapy areshown only for the 75 mg b.i.d. and 125 mg b.i.d. doses compared withthe control arm as randomised. The analysis of the 4 mg b.i.d. dose wasrestricted to subjects randomised to the control arm and receiving 4 mgb.i.d. with or without AChEI/Mem.

TABLE 7 Analyses of ADAS-cog, ADCS-ADL, LVV, TPV and HV in mild AD andmoderate AD with stratification covariate AChEI/Mem as an interactionterm in the model. Treatment with LMTM alone is compared with thecontrol arm as randomised for the 75 mg b.i.d. and 125 mg b.i.d. doses.The analysis of the 4 mg b.i.d. dose was restricted to subjectsrandomised to the control arm and taking or not taking AD-labelledco-medications. Estimate SE 2.50% 97.50% p value ADAS-cog Mild ADControl 2.27 0.77 0.77 3.77 75 mg b.i.d. −8.89 1.76 −12.37 −5.41 5.5e−07125 mg b.i.d. −5.03 1.81 −8.58 −1.47 0.00557 4 mg b.i.d. + 3.24 0.801.67 4.81 AChEI/Mem 4 mg b.i.d. −6.41 1.55 −9.44 −3.37 3.5e−05 ModerateAD Control 7.89 0.68 6.55 9.23 75 mg b.i.d. −2.01 1.94 −5.81 1.80 0.301125 mg b.i.d. −5.75 1.92 −9.51 −1.98 0.00277 4 mg b.i.d. + 8.49 0.717.09 9.88 AChEI/Mem 4 mg b.i.d. −4.74 1.58 −7.83 −1.65 0.00267 ADCS-ADLMild AD Control −3.20 0.96 −5.08 −1.33 75 mg b.i.d. 4.65 2.35 0.04 9.250.0480 125 mg b.i.d. 7.77 2.39 3.10 12.44 0.00112 4 mg b.i.d. + −3.861.01 5.83 −1.88 AChEI/Mem 4 mg b.i.d. 5.20 2.03 1.23 9.17 0.0103Moderate AD Control −10.93 0.95 −12.80 −9.07 75 mg b.i.d. 6.89 2.67 1.6612.13 0.00989 125 mg b.i.d. 5.53 2.66 0.31 10.75 0.0377 4 mg b.i.d. +−11.91 0.99 −13.85 −9.97 AChEI/Mem 4 mg b.i.d. 7.70 2.20 3.39 12.014.6e−04 LVV Mild AD Control 5,789 374 5,056 6,522 75 mg b.i.d. −2,549835 −4,185 −913 0.00226 125 mg b.i.d. −2,695 841 −4,343 −1,047 0.00135 4mg b.i.d. + 6,167 389 5,355 6,879 AChEI/Mem 4 mg b.i.d. −2,655 695−4,021 −1,297 1.3e−04 Moderate AD Control 8,023 372 7,293 8,753 75 mgb.i.d. −2,251 925 −4,065 −438 0.0150 125 mg b.i.d. −1,703 916 −3,499 920.0630 4 mg b.i.d. + 8,143 386 7,386 8,901 AChEI/Mem 4 mg b.i.d. −940777 −2,463 583 0.226 TPV Mild AD Control −1,309 73 −1,452 −1,166 75 mgb.i.d. 392 170 60 726 0.0208 125 mg b.i.d. 439 170 106 772 0.00978 4 mgb.i.d. + −1,398 76 −1,547 −1,249 AChEI/Mem 4 mg b.i.d. 722 144 440 1,0055.5e−07 Moderate AD Control −1,649 59 −1,764 −1,534 75 mg b.i.d. 469 155164 773 0.00257 125 mg b.i.d. 293 154 −9 895 0.0570 4 mg b.i.d. + −1,69761 −1,817 −1,576 AChEI/Mem 4 mg b.i.d. 370 129 116 623 0.00425 HV MildAD Control −115.8 6.6 −126.7 −100.8 75 mg b.i.d. 16.9 16.0 −14.7 48.10.267 125 mg b.i.d. 40.5 16.0 9.2 71.9 0.0113 4 mg b.i.d. + −119.5 6.9−113.1 −106.0 AChEI/Mem 4 mg b.i.d. 46.7 13.9 19.6 73.9 7.5e−04 ModerateAD Control −116.9 4.9 −126.5 −107.3 75 mg b.i.d. 3.6 13.6 −23.1 30.40.789 125 mg b.i.d. 2.2 13.7 −24.8 29.1 0.875 4 mg b.i.d. + −116.6 5.1−126.6 106.6 AChEI/Mem 4 mg b.i.d. −2.4 11.2 −24.4 19.6 0.829

Comparison at Baseline of Patients Taking or Not-Taking AD-LabelledMedications

Given the differences in outcomes according to whether LMTM was taken asmonotherapy or not, differences at baseline between these groups wereanalysed according to clinical severity (Table 8). No difference wasfound in age or sex distribution. There was a significant regionaldifference in mild (but not moderate) patients, in that patients notprescribed AD-labelled co-medications were found to be at sites locatedpredominantly in Russia, Eastern Europe (Poland and Croatia) andMalaysia. There was no difference in baseline ADAS-cog or MMSE. Mildpatients not taking these medications were marginally worse on theADCS-ADL scale, but there was no difference for moderate patients. Interms of the MRI parameters at baseline, mild (but not moderate)patients not taking these medications had a slightly larger HV and LVV,but there was no difference in WBV, TPV or in extent of vascularpathology burden as indicated by Fazekas score at baseline (Murray etal., 2005). Likewise no differences were found for baseline bilirubin orcreatinine clearance would might suggest differences in metabolism orexcretion of LMTM.

TABLE 8 Differences at baseline between patients taking or not takingAD-labelled co-medications. Mild Moderate (not Mild p- (not Moderate p-taking) (taking) value taking) (taking) value Sex 0.142 0.759 Age 70.671.7 0.251 72.2 70.1 0.555 (5.8) (8.3) (9.8) (8.6) Region 0.00763 0.212ADAS-cog 20.1 20.2 0.366 33.2 31.2 0.846 (5.6) (7.1) (9.8) (9.2)ADCS-ADL 60.7 61.6 0.00106 47.7 53.2 0.147 (7.3) (9.7) (16.6) (12.8)MMSE 22.3 22.2 0.705 16.5 16.4 0.585 (1.8) (2.2) (1.7) (1.9) WBV 946,295950,840 0.664 925,032 912,214 0.357 (97,194) (116,924) (111,971)(100,101) TPV 41,841 41,911 0.488 38,208 38,069 0.229 (6,065) (5,733)(5,366) (5,101) HV 3,153 2,775 7.2e−05 2,654 2738 0.0893 (647) (534)(610) (508) LVV 37,975 51,732 0.00196 56,541 53,719 0.600 (17,346)(24,072) (25,660) (23,021) Fazekas score 0.149 0.463 Creatinine 66.067.2 0.701 68.6 65.9 0.332 clearance Bilirubin 0.51 0.56 0.235 0.53 0.520.772

Safety Outcomes

The gastrointestinal and urinary tracts were the body systems mostcommonly affected by adverse events. These were also the most commonreasons for discontinuing high dose LMTM (9% and 3% of patients,respectively); in comparison, only 1-2% of control patients discontinuedfor these events. Of note, the incidence of gastrointestinal adverseevents was about two-fold higher in patients also receivingacetylcholinesterase inhibitors (data not shown). The treatment emergentadverse events occurring in ≥5% on high dose LMTM and greater than inthe control arm are shown in Table 9.

TABLE 9 Most common treatment emergent adverse events occurring in ≥5%on 75 mg b.i.d. or 125 mg b.i.d. LMTM and greater than in control arm.High dose LMTM MedDRA System Control Organ Class/ 4 mg b.i.d. 75 mgb.i.d. 125 mg b.i.d. Preferred term (n = 354) (n = 267) (n = 264) Atleast one TEAE 296 (83.6%) 224 (83.9%) 229 (86.7%) Blood and lymphatic17 (4.8%) 29 (10.9%) 25 (9.5%) system disorders Anemia 10 (2.8%) 22(8.2%) 15 (5.7%) Gastrointestinal 87 (24.6%) 105 (39.3%) 111 (42.0%)disorders Diarrhea 33 (9.3%) 63 (23.6%) 67 (25.4%) Nausea 14 (4.0%) 22(8.2%) 19 (7.2%) Vomiting 2 (0.6%) 25 (9.4%) 18 (6.8%) Infections and 88(24.9%) 83 (31.1%) 76 (28.8%) infestations Urinary tract infection 29(8.2%) 29 (10.9%) 26 (9.8%) Investigations 80 (22.6%) 87 (32.6%) 80(30.3%) Blood folate decreased 21 (5.9%) 18 (6.7%) 19 (7.2%) Renal andurinary 29 (8.2%) 61 (22.8%) 65 (24.6%) disorders Dysuria 3 (0.8%) 7(2.6%) 27 (10.2%) Pollakiuria 6 (1.7%) 15 (5.6%) 18 (6.8%) Urinaryincontinence 9 (2.5%) 18 (6.7%) 12 (4.5%) Respiratory, thoracic 28(7.9%) 32 (12.0%) 22 (8.3%) and mediastinal disorders Cough 12 (3.4%) 14(5.2%) 11 (4.2%)

Adverse events of special interest (AESIs) were identified based on theknown pharmacology of the MT moiety (specifically, ARIA). When thevarious adverse event terms are grouped, anaemia-related terms werereported in 22% of patients receiving high dose LMTM (as compared to 16%receiving control). The maximum mean changes in haemoglobin frombaseline were at 6 weeks and were respectively (in g/L) −0.01 (95% CI:−0.23-0.21, p-value=0.914), −0.47 (95% CI: −0.73-−0.22, p-value=6.4e-04)and −0.93 (95% CI: −1.18-−0.68, p-value=1.2e-12) g/L at doses of 4, 75and 125 mg b.i.d. respectively. There was no case of haemolytic anemia.Twenty two percent of patients entered the study taking an SSRI(selective serotonin reuptake inhibitor). Only 2 patients had transientsymptoms consistent with serotonergic excess but the temporal course andpresentation were not consistent with serotonin toxicity; both patientswere treated with LMTM 75 mg b.i.d. and neither received a concomitantserotonergic drug. In total, 8 patients developed ARIA during the study(<1%), with no dose relationship.

Based on the Columbia Suicide Severity Rating Scale, 26 patients hadtransient responses indicating a wish to be dead. There was one suicideattempt. With respect to other significant events, 9 patients whoparticipated in the study died, the most common reasons beingprogression of AD or cancer; none was related to treatment. A total of97 patients had one or more other non-fatal serious adverse events(SAEs), consistent with the nature of the patient population and evenlydistributed between the three treatment groups. These were possiblyrelated to treatment in only 14% of the cases, the most common beingconvulsion (all 4 occurring in the control arm).

TABLE 10 Significant treatment emergent adverse events Control High doseLMTM 4 mg b.i.d. 75 mg b.i.d. 125 mg b.i.d. Category (n = 354) (n = 267)(n = 264) Deaths, n (%) 3 (0.8) 3 (1.1) 3 (1.1) Adverse Events ofSpecial Interest (AESIs) Methemoglobinemia, 1 (0.3) 0 (0) 1 (0.4)hemolytic anemia, and/or Heinz bodies, n (%) “Serotonin syndrome”, 0 (0)2 (0.7) 0 (0) n (%) ARIA, n (%) 3 (0.8) 4 (1.5) 1 (0.4) Other SUSARs, n(%) 2 (0.6) 2 (0.7) 4 (1.5)

Discussion

The purpose of the present study was to confirm the efficacy reported inthe earlier phase 2 study using a total daily dose of 150 mg/day and todetermine whether 250 mg/day could achieve superior benefit using anewly developed stabilised reduced form of MT as LMTM. The study used alow dose of LMTM (4 mg b.i.d.) in the control arm rather than a trueplacebo to ensure blinding with respect to discolouration of excreta.The rates of decline on the ADAS-cog and ADCS-ADL scales seen in thecontrol arm were linear and indistinguishable from those reported inrecent studies. The same was found to be true for the rate ofprogression of brain atrophy in the mild AD group measured by change ofLVV in comparison with data available from the ADNI program (Frisoni etal., 2010; Nestor et al., 2008) These comparisons support the facevalidity of the present study.

The AChEI/Mem factor was defined as a stratification variable, alongwith baseline severity and geographical region, thereby ensuring thatthe randomised treatment arms were equally represented in all threestrata. It was assumed that it would be sufficient to account for theAChEI/Mem effect by including it as an additive term in the model, alongwith the other stratification factors. The primary efficacy analysis asprespecified did not, however, demonstrate statistical significance oneither of the primary efficacy outcomes at either 75 mg b.i.d. or 125 mgb.i.d. using this model. The same analysis showed that the AChEI/Memfactor was a statistically significant determinant of efficacy, suchthat those not taking AD-labelled drugs experienced a mean overallbenefit relative to controls of −2.30 ADAS-cog units and 2.00 ADCS-ADLunits, effects that remained statistically significant in thewhole-population analysis after full correction for multiplecomparisons. Since the baseline values were by definition zero, such anoverall benefit could only occur if the intended active treatmentsproduced a difference in the rate of progression relative to controls inpatients taking LMTM as monotherapy.

To confirm this, the effect of LMTM treatment in the whole populationwas re-examined using a prespecified analysis model in which theAChEI/Mem term was included as an interaction term with visit and aninteraction term with LMTM treatment, rather than only as an additiveterm. This analysis confirmed that treatment benefit was restricted topatients taking LMTM as monotherapy. LMTM at doses of 75 mg b.i.d. or125 mg b.i.d. produced treatment effects of −6.25 and −5.79 ADAS-cogunits respectively at 65 weeks, or 103%±23% and 83%±23% (mean±SE) of thedecline over 65 weeks seen in the control arm. The corresponding effectsizes on the ADCS-ADL scale were 6.48 and 6.92, or 82%±23% and 88%±23%of the decline seen over 65 weeks in the control arm. An identicalprofile was found for MRI measures of progression of neocorticalatrophy, with reductions of 38%±9% and 33%±9% in LVV and increases of31%±8% and 26%±8% in TPV for the 75 mg b.i.d. and 125 mg b.i.d. doses.All of these effects were statistically robust after appropriatecorrection for multiple comparisons. By contrast, the decline seen atthe same doses in patients taking LMTM in combination with AD-labelledtreatments, who were the majority, was indistinguishable on allparameters from that seen in the control arm.

Given that the higher dose did not result in greater efficacy, weexamined whether differential efficacy for LMTM as monotherapy or notmight also be present at the 4 mg b.i.d. dose originally intended as aurinary and faecal discolourant. A post hoc analysis showed that 4 mgb.i.d. as monotherapy showed effects of −5.90 ADAS-cog units, 7.19ADCS-ADL units, as well as benefits in LVV and TPV similar to those seenat the higher doses relative to patients taking the same dose incombination with standard AD treatments, and in whom the decline wasagain indistinguishable from the decline seen either in the control armsof recently reported studies or ADNI data.

The efficacy profiles were also similar in mild and moderate subjects.The only difference was that, in hippocampus, benefit was seen in mildpatients (increased by 35.5 mm³) but not in moderate patients. This isconsistent with the known staging of tau aggregation pathology, wherebydamage in medial temporal lobe structures occurs earlier and is moresevere than in neocortex. The general concordance of benefit on thevolumetric measures of rate of progression of brain atrophy by LVV, TPVand HV, particularly in mild AD, argues against the possibility that theLVV measure is simply reporting treatment-related fluid shifts.Therefore TAI therapy has potential to benefit patients at both the mildand moderate stages of the disease, and not just at more advanced stagesof AD as has been supposed. Indeed, the mean treatment benefit for LMTMmonotherapy relative to control was 240%±41% and 156%±39% on theADAS-cog and ADCS-ADL in mild patients.

The benefit seen with LMTM monotherapy at doses of 4, 75 and 125 mgb.i.d. is comparable to that seen on the ADAS-cog scale at 12 months inthe phase 2 study where MTC was also given as monotherapy at 47 mg MTt.i.d. (Wischik et al., 2015). We have recently reported that theabsorption and distribution of MT to the brain is complex, and likely tobe mediated via red cells rather than plasma, (Baddeley et al., 2015)providing a route which protects MT from first-pass metabolism. In thesame study MT uptake into red cells was approximately 20-fold higher invivo when as administered intravenously as LMTM compared with MTC, mostlikely due to direct red cell uptake of LMT by passive diffusion withoutneed for prior reduction of MT⁺ as is the case for MTC (Baddeley et al.,2015; May et al., 2004). The results of the present study suggest thatMT uptake and distribution are capacity-limited by the amount that redcells can take up whilst within the portal circulation.

The reason for the loss of benefit on clinical and volumetric outcomeswhen LMTM is combined with symptomatic AD treatments remains to beexplained, and studies are ongoing which aim to understand this better.To date, an interference at the site of action at the high affinitytau-tau binding site has been ruled out in vitro in both cell-free andcellular assays (Harrington et al., 2015). Likewise a direct effect ondissolution of LMTM tablets or complexing of LMTM with the ADmedications or their excipients has been ruled out (unpublished data).The interference does not occur in certain other neurodegenerativedisorders of protein aggregation (unpublished data), implying that theinterference effect shown in AD is not applicable to all MT treatmentsof neurodegenerative disorders and may indeed be disease-specific. Onepossible contributory factor may be induction of the multidrugresistance protein 1 (MDR1), a transporter which is upregulated byAChEIs and memantine (Mohamed et al., 2015, 2016). We have shown that MTis a pH-dependent substrate for this pathway (unpublished data). The neteffect could be enhanced efflux of MT from the brain, enhanced liveruptake leading to conjugation and inactivation of MT and faecalexcretion, and also enhanced excretion of MT via the kidney. This maylower the concentration of MT at the site of action below a criticallevel required for efficacy in AD. Further studies to confirm this orother hypotheses are in progress.

The overall safety of LMTM as monotherapy is consistent with priorexperience with MTC (Wischik et al., 2015). Adverse events affecting thegastrointestinal and urinary systems were the most common, and were alsothe most common reason for discontinuing high dose LMTM. Reporting ofreductions in red cell indices was greater in patients receiving higherdoses of LMTM, consistent with effects previously described for MTC,(Baddeley et al., 2015) although there was no significant reduction inhaemoglobin at the 4 mg b.i.d. dose. Although 22% of patients weretaking SSRIs, only 2 patients had transient symptoms meeting any of thecriteria for serotonin toxicity, but neither of these was taking an SSRI(or any other serotonergic drug). Out of the 9 deaths that occurredduring the study, none was related to treatment. Eight developed ARIAduring the study (<1%) but there was no dose relationship. Thisfrequency is consistent with the placebo rates reported in recent trials(Doody et al., 2014).

In conclusion, the results herein demonstrate the potential benefits ofadding a tau-based approach to those currently available or planned forthe treatment of diseases such as mild and moderate AD. A dose of LMTMas low as 4 mg b.i.d. as monotherapy may be the optimal dose in mild AD,able to produce substantial clinical benefits whilst being welltolerated and having fewer side effects than the higher doses. Suchtreatment would need to be introduced either prior to or followingcessation of the currently available AD treatments, as the combinationappears to eliminate benefit.

Example 4—Further Phase 3 Clinical Trial in Mild AD

Objectives

To examine the potential efficacy of LMTM as monotherapy innon-randomised observational cohort analyses as modified primaryoutcomes in an 18-months Phase 3 trial in mild AD.

Methods

Mild AD patients (n=800) were randomly assigned to 100 mg twice a day or4 mg twice a day.

The Statistical Analysis Plan was revised in light of Example 3 (whichcompleted earlier) prior to database lock and unblinding, to compare the100 mg twice a day as monotherapy subgroup (n=79) versus 4 mg twice aday as randomised (n=396), and 4 mg twice a day as monotherapy (n=76)versus 4 mg twice a day as add-on therapy (n=297), with strong controlof family-wise type I error.

Results

The revised analyses were statistically significant at the requiredthreshold of p<0.025 in both comparisons on the co-primary clinicalefficacy endpoints (ADAS-cog and ADCS-ADL), MRI atrophy and glucoseuptake. Whole brain atrophy progressed initially as expected for mild ADin both add-on and monotherapy groups, but diverged significantly after9 months of treatment, with the final atrophy rate in monotherapypatients typical of normal elderly controls. Differences at baselinebetween monotherapy and add-on patients did not account for significantdifferences in favour of monotherapy. Treatment response to LMTM asadd-on was inversely correlated with relative basal forebrain atrophy.

CONCLUSIONS

The as-randomised analyses of two Phase 3 trials using LMTM aredescribed herein: the first in mild to moderate AD (Example 3) and thesecond in mild AD (this Example). Both studies were originally designedto compare higher doses of LMTM in the range 150-250 mg/day with a lowdose of 4 mg twice a day intended as a urinary discolourant to maintainblinding. It was assumed that this low dose would be ineffective, sincea dose of 69 mg MT/day as MTC was found to have minimal efficacy in thePhase 2 study.

Neither Phase 3 trial study showed any difference on primary orsecondary outcomes between the high doses and 4 mg twice a day. In thefirst study (Example 3) treatment status with cholinesterase inhibitorsand/or memantine was found to be a significant covariate in the primaryanalysis model. Exploratory analyses showed that this was due tosignificantly lower rates of progression on clinical and brain atrophyendpoints in patients receiving any of the LMTM doses as monotherapy,including 4 mg twice a day, which did not appear to be explicable bycohort differences in severity at baseline.

The results of the Example 3 study raised the possibility LMTM might bemost effective as a monotherapy and that the minimum effective dosemight be substantially lower for LMTM than that previously expected forMTC (see e.g. WO2009/044127).

We therefore modified the primary analyses and treatment comparisons inthe study described in this Example (prior to database lock andunblinding) to investigate whether the monotherapy differences could beconfirmed as observational cohort comparisons defined as primaryoutcomes with strong control of family-wise type I error in the secondindependent study. The monotherapy cohort comparisons which were ofparticular interest in light of the earlier study were: (A) 100 mg twicea day a monotherapy compared with the controls as originally randomised,and (B) 4 mg twice a day as monotherapy compared with the same dose asadd-on to standard AD treatments.

Both primary Comparisons A and B met the required statistical thresholdof p<0.025 for both co-primary clinical outcomes (ADAS-cog andADCS-ADL), as well as for volumetric MRI and glucose uptake biomarkeroutcomes. Specifically, patients receiving LMTM as monotherapy at eitherof the two doses tested had consistently better outcomes than patientsreceiving the same doses as add-on to cholinesterase inhibitors and/ormemantine.

This confirmation of the same pattern of results in this second,independent, study argues against either the present findings or thosereported as post hoc findings from the earlier mild/moderate AD study ofExample 3 being the result of chance in small subgroups, although themonotherapy subgroups remain small in the present study (155 or 20% intotal in the mITT analyses).

It is also unlikely that the earlier findings of Example 3 areexplicable by inclusion of non-western geographies, since the presentstudy was conducted in north America, western Europe and Australia. Aclinical placebo effect in patients coming into a trial setting afterpreviously not receiving active treatment cannot explain the samepattern of results seen in both the MRI brain atrophy and ¹⁸F-FDG-PETfunctional data as seen in the clinical data. A difference in withdrawalrates between patients taking or not taking standard AD treatments isalso unlikely, since the overall retention rates over 18 months weresimilar in monotherapy (65%) and add-on (69%) treatment groups.

The pattern of atrophy at baseline in patients receiving LMTM asmonotherapy was typical of mild AD and significantly different from acohort of well characterised normal elderly controls. The annualisedrate of whole brain atrophy in these patients over the first 6 monthswas also similar to that reported for mild AD and significantlydifferent from normal elderly controls. Likewise glucose uptake ininferior temporal gyrus was comparable in monotherapy patients to thatreported for mild AD and significantly different from MCI or normalelderly controls. In addition to meeting clinical diagnostic criteriafor mild AD, the baseline imaging data therefore confirm that thepatients not prescribed cholinesterase inhibitors or memantine can betaken as typical of mild AD.

Patients not receiving standard AD treatments were somewhat lessimpaired at study entry on the ADAS-cog, ADCS-ADL, MMSE scales, as wellas in ventricular, temporoparietal and hippocampal atrophy, and temporallobe glucose uptake. It is therefore possible that this difference inseverity at baseline might have accounted for significant differences inprogression. However, baseline severity was included as an additive termin the primary analysis models and was therefore corrected for. Wefurther tested whether baseline severity or other patientcharacteristics could explain differences in rate of progression byundertaking sensitivity analyses with additional rate-correction termsin the analysis model. If differences in baseline characteristicsexplain the differences seen over 18 months, then the significantdifferences in favour of LMTM as monotherapy would be expected todisappear when corrected for baseline effects, as they did in a similaranalysis examining differences in rate of progression according to ADtreatment status in patients with MMSE 20-26 in the currently availableADNI data set (unpublished observation). Rate-correction for differencesin clinical severity at baseline, APO ε4 frequency, vascular pathologyload, hippocampal atrophy, temporoparietal atrophy and glucose uptake inthe temporal lobe did not eliminate the significant differences infavour of LMTM monotherapy for ADAS-cog, ADCS-ADL or lateral ventricularvolume. We further examined whether the differences in favour of LMTM asmonotherapy depend on inclusion of patients receiving a cholinesteraseinhibitor and memantine in combination, which could be taken to reflecta potential prescriber perception of risk of more rapid decline.Removing them had minimal effect on the estimates or significance of thetreatment differences. It therefore appears unlikely that the relativelyminor differences in severity or the other characteristics at baselineexplain the significant outcome differences in favour of LMTMmonotherapy over 18 months.

An analysis that is free of between-cohort confounding effects is thewithin-cohort comparison of annualised rate of whole brain atrophy atstudy entry and after 9 months of treatment with LMTM. We found that inpatients receiving LMTM as monotherapy there was a significant delayedreduction in the annualised rate of whole brain atrophy. As noted above,monotherapy patients entered the study with an initial whole brainatrophy rate typical of mild AD and significantly different from thatreported for normal elderly controls. After receiving LMTM asmonotherapy for 9 months, the rate was reduced to that reported fornormal elderly controls and significantly different from mild AD. Thesechanges were not seen in the patients receiving LMTM as add-on therapy,who continued to decline as expected for mild AD. Similarly, the declinein temporal lobe glucose uptake in patients receiving LMTM asmonotherapy was significantly less than reported for mild AD.

Decline on the ADAS-cog scale in patients receiving LMTM in combinationwith a cholinesterase inhibitor was found to vary inversely with atrophyin the nucleus basalis and nucleus accumbens relative to whole brainvolume. A similar effect was also seen for cortical glucose uptake. Thecorresponding effect of basal forebrain atrophy was weak or absent forthe LMTM/memantine combination and was also absent for LMTM monotherapy.Both of these basal forebrain nuclei are known to be affected by tauaggregation pathology. The role of nucleus basalis in determiningtreatment response may help to provide some insight into a possiblemechanism underlying the negative interaction with cholinesteraseinhibitors. Ascending cholinergic projections originating predominantlyfrom nucleus basalis provide both direct activation and indirectinhibitory modulation of cortical pyramidal cells (Huang M, Felix A R,Flood D G, Bhuvaneswaran C, Hilt D, Koenig G, Meltzer H Y (2014) Thenovel α7 nicotinic acetylcholine receptor agonist EVP-6124 enhancesdopamine, acetylcholine, and glutamate efflux in rat cortex and nucleusaccumbens. Psychopharmacology 231, 4541-4551; Picciotto Marina R, HigleyMichael J, Mineur Yann S (2012) Acetylcholine as a Neuromodulator:Cholinergic Signaling Shapes Nervous System Function and Behavior.Neuron 76, 116-129). Memantine also increases release of acetyl cholinein nucleus accumbens (Shearman E, Rossi S, Szasz B, Juranyi Z, Fallon S,Pomara N, Sershen H, Lajtha A (2006) Changes in cerebralneurotransmitters and metabolites induced by acute donepezil andmemantine administrations: A microdialysis study. Brain Res Bull 69,204-213) which modulates cortical activity indirectly. Long-terminhibition of cholinesterase activity combined with loss of inhibitorymodulation may therefore result in chronic hyperactivation of pyramidalcells in cortex (and in CA 1-3 of hippocampus) which are the principalsites of neurofibrillary degeneration in AD (Lewis D A, Campbell M J,Terry R D, Morrison J H (1987) Laminar and regional distributions ofneurofibrillary tantles and neuritic plaques in Alzheimer's disease: aquantitative study of visual and auditory cortices. J Neurosci 7,1799-1808). It is therefore possible that the relative severity of basalforebrain pathology together with chronic choline esterase inhibitionmay determine the degree of hyperactivation of cortical pyramidal cellsand that this impairs the action of MT even at high dose. We show thatwhereas the treatment response to LMTM as add-on to approved treatmentsfor AD varies according to the severity of relative basal forebrainatrophy, the effect of LMTM as monotherapy does not. The difference intreatment response between LMTM monotherapy and add-on therapy cannottherefore be attributed simply to cohort differences between patientsprescribed or not prescribed such treatments. It also cannot beattributed to relative lack of pathology, since patients with thegreatest basal forebrain atrophy responded significantly better tomonotherapy than to combination treatment.

The potential for LMTM to be active at the low dose of 4 mg twice a dayand the lack of dose-response are at first sight surprising given theresults of an earlier Phase 2 placebo-controlled study using theoxidised form of the methylthioninium (MT) moiety as methylthioniniumchloride (MTC) (Wischik, 2015). The stable reduced form of MT (as LMTM)was developed to overcome the absorption limitations observed for MTC.LMTM has 20-fold better red cell uptake than MTC in vivo and also betterbrain uptake. Recent studies in rodents dosed orally with LMTM tosimulate the 4 mg twice a day dose in humans found brain levels of MT tobe on 0.1-0.2 μM, similar to the steady state concentration estimatedfor the minimum effective dose of MTC. A concentration of approximately0.05 μM appears to be adequate for a range of reported potentiallybeneficial effects of the MT moiety such as enhancement of autophagy andenhancement of mitochondrial function. The concentration required fordissolution of PHFs and oligomers in vitro is approximately 1/10^(th)that of aggregated tau, implying that a concentration of 0.05 μM may beadequate in vivo, given the brain concentrations of aggregated tau thathave been reported. There is no dose-response for oligomerdisaggregation in vitro, and higher doses of LMTM did not result ingreater reduction in tau pathology in transgenic mouse models, at leastin the range tested (Melis, 2015) Effects of oxidized and reduced formsof methylthioninium in two transgenic mouse tauopathy models. BehavPharmacol 26, 353-368). This suggests that there may be a criticalthreshold for activity at the tau aggregation inhibitor target, and theeffect of higher doses may plateau or may even become negative at brainconcentrations above 1 μM (Melis, 2015). Several results suggest that 4mg twice a day may serve better than 100 mg twice a day. The clinicaldifferences in favour of 4 mg twice a day were seen at both CDR 0.5 and1.0, but only at CDR 0.5 at the higher dose, and the glucose uptakedifference in temporal cortex occurred earlier at the lower dose.

The lower dose of 4 mg twice a day had a better overall clinical profilethan 100 mg twice a day. The withdrawal rate over 18 months for the 4 mgtwice a day dose was less (25%, 94/296) than at 100 mg twice a day (46%,182/399), and the adverse event profile was more benign with respect tothe diarrhoea, dysuria and decreased haemoglobin. There is no increasedrisk of cerebral microhaemorrhages or oedema with LMTM even at thehigher dose, since the ARIA rates observed in both Phase 3 studiesreported herein were similar to those previously reported for placebocontrols (Doody, 2014; Salloway, 2014).

The differences in favour of LMTM as monotherapy are based onobservational cohort analyses, albeit defined a priori as statisticallyprimary outcomes for the modified analysis we report here. This patternof results has been seen now in both separate Phase 3 studies, implyingthat the effects are consistent across studies. The differencesfavouring monotherapy are also internally consistent across a range ofclinical outcomes, and the clinical outcomes are consistent with theneuroimaging outcomes in both studies.

Allowing for differences in absorption between LMTM and MTC, the resultsare also consistent with the earlier Phase 2 placebo-controlled studysupporting potential efficacy of the MT moiety as monotherapy, andunderline the potential beneficial clinical and biological effects ofLMTM as monotherapy at the safe and well-tolerated dose of around 4 mgtwice a day.

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The invention claimed is:
 1. A method of therapeutic treatment of mild cognitive impairment in a subject, which method comprises orally administering to said subject a methylthioninium (MT)-containing compound, wherein said administration provides a total daily dose of between 0.5 and 20 mg of MT to the subject per day, wherein the MT-containing compound is LMTM:


2. The method as claimed in claim 1, wherein the total daily dose of MT is 2 to 15 mg.
 3. The method as claimed in claim 1, wherein said therapeutic treatment with the MT-containing compound comprises a total daily dose of LMTM of 0.8 to 33 mg/day.
 4. The method as claimed in claim 1, wherein said therapeutic treatment with the MT-containing compound comprises a dose of LMTM of 9 mg/once per day.
 5. A method of prophylactic treatment of mild cognitive impairment in a subject, which method comprises orally administering to said subject a methylthioninium (MT)-containing compound, wherein said administration provides a total daily dose of between 0.5 and 20 mg of MT to the subject per day, wherein the MT-containing compound is LMTM:


6. The method as claimed in claim 1, wherein the subject has not historically received treatment with an acetylcholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist.
 7. The method as claimed in claim 1, wherein the subject has historically received treatment with an acetylcholinesterase inhibitor and\or an N-methyl-D-aspartate receptor antagonist, but ceased that medication at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks prior to treatment with the MT containing compound.
 8. The method as claimed in claim 1, wherein the subject is selected as one who is receiving treatment with an acetylcholinesterase inhibitor and\or an N-methyl-D-aspartate receptor antagonist, wherein said treatment with the acetylcholinesterase inhibitor and\or an N-methyl-D-aspartate receptor antagonist is discontinued prior to treatment with the MT-containing compound.
 9. The method as claimed in claim 1, wherein said therapeutic treatment comprises a total daily dose of the MT-containing compound administered once per day.
 10. The method as claimed in claim 1, wherein said therapeutic treatment comprises a total daily dose of the MT-containing compound administered as a split dose twice per day.
 11. The method as claimed in claim 1, wherein said therapeutic treatment comprises a total daily dose of the MT-containing compound administered as a split dose three times per day.
 12. The method as claimed in claim 5, wherein said prophylactic treatment comprises a total daily dose of the MT-containing compound administered once per day.
 13. The method as claimed in claim 5, wherein said prophylactic treatment comprises a total daily dose of the MT-containing compound administered as a split dose twice per day.
 14. The method as claimed in claim 5, wherein said prophylactic treatment comprises a total daily dose of the MT-containing compound administered as a split dose three times per day.
 15. The method as claimed in claim 2, wherein the total daily dose of MT is 3 to 10 mg.
 16. The method as claimed in claim 3, wherein the total daily dose of LMTM is 6 to 12 mg/day. 